Optimized antibodies that target CD19

ABSTRACT

The present invention describes antibodies that target CD19, wherein the antibodies comprise at least one modification relative to a parent antibody, wherein the modification alters affinity to an FcgγR or alters effector function as compared to the parent antibody. Also disclosed are methods of using the antibodies of the invention.

This application is a division of allowed U.S. patent application Ser.No. 12/377,251, filed Feb. 11, 2009, which is the national stageapplication of PCT Patent Application No. PCT/US2007/75932, filed Aug.14, 2007, and which claims benefit under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 60/822,362, filed Aug. 14, 2006, allof which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

A Sequence Listing submitted in computer readable form (CRF) is herebyincorporated by reference. The CRF file is named 189519.ST25.txt, wascreated on Aug. 30, 2011, and contains 190 kilobytes.

BACKGROUND

B Cells

B cells are lymphocytes that play a large role in the humoral immuneresponse. They are produced in the bone marrow of most mammals, andrepresent 5-15% of the circulating lymphoid pool. The principal functionof B cells is to make antibodies against various antigens, and are anessential component of the adaptive immune system.

The human body makes millions of different types of B cells each daythat circulate in the blood and lymph performing the role of immunesurveillance. B cells, also referred to as B lymphocytes, do not produceantibodies until they become fully activated. Each B cell has a uniquereceptor protein (referred to as the B cell receptor (BCR)) on itssurface that will bind to one particular antigen. The BCR is amembrane-bound immunoglobulin, and it is this molecule that allows thedistinction of B cells from other types of lymphocytes, as well as beingthe main receptor involved in B-cell activation. Once a B cellencounters its cognate antigen and receives an additional signal from aT helper cell, it can further differentiate into various types of Bcells listed below. The B cell may either become one of these cell typesdirectly or it may undergo an intermediate differentiation step, thegerminal center reaction, where the B cell will hypermutate the variableregion of its immunoglobulin gene and possibly undergo class switching.

B-cell development occurs through several stages, each stagerepresenting a change in the genome content at the antibody loci. Thestages of B-cell development include Progenitor B cells, Early Pro-Bcells, Late Pro-B cells, Large Pre-B cells, Small Pre-B cells, ImmatureB cells, and Mature B cells.

Mature B cells can be divided into four major types:

B-1 cells express CD5, a marker usually found on T cells. B-1 cells alsoexpress IgM in greater quantities than IgG. They secrete natural lowaffinity polyreactive antibodies found in the serum and often havespecificities directed toward self-antigens, and common bacterialpolysaccharides. B-1 cells are present in low numbers in the lymph nodesand spleen and are instead found predominantly in the peritoneal andpleural cavities.

B-2 cells are the conventional B cells to which most texts refer. Theyreside in bone marrow, spleen, and lymph nodes. They are short-lived,and when triggered by antigens may differentiate into IgG-producingmemory B cells. In the course of these antibody responses IgG mayundergo substantial affinity maturation.

Plasma B cells (also known as plasma cells) are large B cells that havebeen exposed to antigen and produce and secrete large amounts ofantibodies, which assist in the destruction of microbes by binding andfacilitating targeting by phagocytes, as well as activation of thecomplement system. Plasma cells are sometimes referred to as antibodyfactories.

Memory B cells are formed from activated B cells that are specific tothe antigen encountered during the primary immune response. These cellslive for a long time, and can respond quickly following a secondexposure to the same antigen.

When a B cell fails in any step of the maturation process, it will dieby a mechanism called apoptosis. If it recognizes self-antigen duringthe maturation process, the B cell will become suppressed (known asanergy) or undergo apoptosis. B cells are continuously produced in thebone marrow, but only a small portion of newly made B cells survive toparticipate in the long-lived peripheral B-cell pool.

In recent years, data have emerged suggesting that B lymphocytes play abroader role in immune responses and are not merely the passiverecipients of signals that result in differentiation intoantibody-producing plasma cells. Along with their traditional roles asantigen presenting cells and precursors of antibody-producing plasmacells, B cells have also been found to regulate antigen presenting cells(APCs) and T-cell functions, produce cytokines, and expressreceptor/ligand pairs that previously had been thought to be restrictedto other cell types.

B-cell Disorders

Because of their critical role in regulating the immune system,disregulation of B cells is associated with a variety of disorders.B-cell disorders, also referred to herein as B-cell related diseases,are divided into excessive or uncontrolled proliferation (lymphomas,leukemias), and defects of B-cell development/immunoglobulin production(immunodeficiencies). The majority (80%) of lymphoma cases are of B-cellorigin. These include non-Hodgkin's lymphoma (NHL), acute lymphoblasticleukemia (ALL), and autoimmune related diseases.

NHL is a heterogeneous malignancy originating from lymphocytes. In theUnited States (U.S.), the incidence is estimated at 65,000/year withmortality of approximately 20,000 (American Cancer Society, 2006; andSEER Cancer Statistics Review). The disease can occur in all ages, theusual onset begins in adults over 40 years, with the incidenceincreasing with age. NHL is characterized by a clonal proliferation oflymphocytes that accumulate in the lymph nodes, blood, bone marrow andspleen, although any major organ may be involved.

The diagnosis and histologic characterization of NHL is made using acombination of morphologic and immunophenotype criteria. The currentclassification system used by pathologists and clinicians is the WorldHealth Organization (WHO) Classification of Tumours, which organizes NHLinto precursor and mature B-cell or T-cell neoplasms. The PDQ iscurrently dividing NHL as indolent or aggressive for entry into clinicaltrials. For consistency the present document will also use a similardivision. The indolent NHL group is comprised primarily of follicularsubtypes, small lymphocytic lymphoma, MALT, and marginal zone; indolentencompasses approximately 50% of newly diagnosed B-cell NHL patients.Aggressive NHL includes patients with histologic diagnoses of primarilydiffuse large B cell (40% of all newly diagnosed patients have diffuselarge cell), Burkitt's, and mantle cell.

The clinical course of NHL is highly variable. A major determinant ofclinical course is the histologic subtype. Most indolent types of NHLare considered to be incurable disease. Patients respond initially toeither chemotherapy or antibody therapy and most will relapse. Studiesto date have not demonstrated an improvement in survival with earlyintervention. In asymptomatic patients, it is acceptable to “watch andwait” until the patient becomes symptomatic or the disease pace appearsto be accelerating. Over time, the disease may transform to a moreaggressive histology. The media survival is 8 to 10 years, and indolentpatients often receive 3 or more treatments during the treatment phaseof their disease. Initial treatment of the symptomatic indolent NHLpatient historically has been combination chemotherapy. The mostcommonly used agents include: cyclophosphamide, vincristine andprednisone (CVP); cyclophosphamide, adriamycin, vincristine, prednisone(CHOP); or the purine analog, fludarabine. Approximately 70% to 80% ofpatients will respond to their initial chemotherapy, duration ofremissions last on the order of 2-3 years. Ultimately the majority ofpatients relapse. The discovery and clinical use of the anti-CD20antibody, rituximab, has provided significant improvements in responseand survival rate. The current standard of care for most patients isrituximab+CHOP (R-CHOP) or rituximab+CVP (R-CVP). Interferon is approvedfor initial treatment of NHL in combination with alkylating agents, buthas limited use in the U.S.

Rituximab therapy has been shown to be efficacious in several types ofNHL, and is currently approved as a first line treatment for bothindolent (follicular lymphoma) and aggressive NHL (diffuse large B celllymphoma). However, there are significant limitations of anti-CD20monoclonal antibody (mAb), including primary resistance (50% response inrelapsed indolent patients), acquired resistance (50% response rate uponre-treatment), rare complete response (2% complete response rate inrelapsed population), and a continued pattern of relapse. Finally, manyB cells do not express CD20, and thus many B-cell disorders are nottreatable using anti-CD20 antibody therapy. Antibodies against antigensother than CD20 may have anti-lymphoma effects that could overcomeanti-CD20 resistance or augment the activity of anti-CD20 therapy.

In addition to NHL there are several types of leukemias that result fromdisregulation of B cells. Chronic lymphocytic leukemia (also know as“chronic lymphoid leukemia” or “CLL”), is a type of adult leukemiacaused by an abnormal accumulation of B lymphocytes. In CLL, themalignant lymphocytes may look normal and mature, but they are not ableto cope effectively with infection. CLL is the most common form ofleukemia in adults. Men are twice as likely to develop CLL as women.However, the key risk factor is age. Over 75% of new cases are diagnosedin patients over age 50. More than 10,000 cases are diagnosed every yearand the mortality is almost 5,000 a year (American Cancer Society, 2006;and SEER Cancer Statistics Review).

CLL is an incurable disease but progresses slowly in most cases. Mostpeople with CLL lead normal and active lives for many years. Because ofits slow onset, early-stage CLL is generally not treated since it isbelieved that early CLL intervention does not improve survival time orquality of life. Instead, the condition is monitored over time. InitialCLL treatments vary depending on the exact diagnosis and the progressionof the disease. There are dozens of agents used for CLL therapy.Although the purine analogue fludarabine was shown to give superiorresponse rates than chlorambucil as primary therapy, there is noevidence that early use of fludarabine improves overall survival.Combination chemotherapy regimens such as fludarabine withcyclophosphamide, FCR (fludarabine, cyclophosphamide and rituximab) andCHOP are effective in both newly-diagnosed and relapsed CLL. Allogeneicbone marrow (stem cell) transplantation is rarely used as a first-linetreatment for CLL due to its risk.

“Refractory” CLL is a disease that no longer responds favorably totreatment. In this case more aggressive therapies, including bone marrow(stem cell) transplantation, are considered. The monoclonal antibodyalemtuzumab, directed against CD52, may be used in patients withrefractory, bone marrow-based disease.

Another type of leukemia is acute lymphoblastic leukemia (ALL), alsoknown as acute lymphocytic leukemia. ALL is characterised by theoverproduction and continuous multiplication of malignant and immaturewhite blood cells (also known as lymphoblasts) in the bone marrow.‘Acute’ refers to the undifferentiated, immature state of thecirculating lymphocytes (“blasts”), and that the disease progressionrapidly with life expectancy of weeks to months if left untreated. ALLis most common in childhood with a peak incidence of 4-5 years of age.Children of age 12-16 die more easily from it than others. Currently, atleast 80% of childhood ALL are considered curable. Under 4,000 cases arediagnosed every year and the mortality is almost 1,500 a year (AmericanCancer Society, 2006; and SEER Cancer Statistics Review).

Autoimmunity results from a breakdown of self-tolerance involvinghumoral and/or cell-mediated immune mechanisms in. Among of theconsequences of failure in central and/or peripheral tolerance, aresurvival and activation of self-reactive B cells and T cells. Examplesof autoimmune diseases include, for example, rheumatoid arthritis (RA),systemic lupus erythematosas (SLE or lupus), multiple sclerosis,Sjogren's syndrome, and idiopathic thrombocytopenia purpura (ITP). Thepathogenesis of most autoimmune diseases is coupled to the production ofautoantibodies against self antigens, leading to a variety of associatedpathologies. Autoantibodies are produced by terminally differentiatedplasma cells that are derived from naïve or memory B cells. Furthermore,B cells can have other effects on autoimmune pathology, asantigen-presenting cells (APCs) that can interact with and stimulatehelper T cells, further stimulating the cycle of anti-self immuneresponse. Depletion of B cells can have direct impact on the productionof autoantibodies. Indeed, treatment of RA and SLE with B-cell depletiontherapies such as Rituxan has been demonstrated to have clinical benefitfor both disease classes (Edwards & Cambridge, Nat. Rev. Immunol. 2006;Dass et al., Future Rheumatol. 2006; Martin & Chan, Annu. Rev. Immunol.2006).

Unfortunately, it is not known a priori which mechanisms of action maybe optimal for a given target antigen. Furthermore, it is not knownwhich antibodies may be capable of mediating a given mechanism of actionagainst a target cell. In some cases a lack of antibody activity, eitherFv-mediated or Fc-mediated, may be due to the targeting of an epitope onthe target antigen that is poor for mediating such activity. In othercases, the targeted epitope may be amenable to a desired Fc-mediated orFc-mediated activity, yet the affinity (affinity of the Fv region forantigen or affinity of the Fc region for Fc receptors) may beinsufficient. Towards addressing this problem, the present inventiondescribes modifications to anti-CD19 antibodies that provide optimizedFv- and Fc-mediated activities. A broad array of applications of theseoptimized antibodies are contemplated.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to an antibody bindsCD19, wherein said antibody comprises at least one modification in theconstant region relative to a parent antibody. In a preferredembodiment, the antibody of the invention binds with altered affinity toan Fc receptor or alters effector function as compared to the parentantibody.

In one aspect, the invention is directed to antibody that binds CD19,including at least one modification in the constant region relative to aparent anti-CD19 antibody, wherein the antibody binds with increasedaffinity to the FcγRIIIa receptor as compared to the parent antibody.

In certain aspects, the modification is an amine acid. The modificationcan be at a position selected from the group consisting of 221, 222,223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 243, 244, 245, 246, 247, 249, 255, 257, 260, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278,280, 281, 281, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295,296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 313, 317, 318, 320,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, and 337, wherein numbering is according to the EU index. The aminoacid modification can be a substitution selected from the groupconsisting of 221K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E,225K, 225W, 227E, 227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E,230G, 230Y, 231E, 231G, 231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 233A,233D, 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S,233T, 233V, 233W, 233Y, 234A, 234D, 234E, 234F, 234G, 234H, 234I, 234K,234M, 234N, 234P, 234Q, 234R, 234S, 234T, 234V, 234W, 234Y, 235A, 235D,235E, 235F, 235G, 235H, 235I, 235K, 235M, 235N, 235P, 235Q, 235R, 235S,235T, 235V, 235W, 235Y, 236A, 236D, 236E, 236F, 236H, 236I, 236K, 236L,236M, 236N, 236P, 236Q, 236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E,237F, 237H, 237I, 237K, 237L, 237M, 237N, 237P, 237Q, 237R, 237S, 237T,237V, 237W, 237Y, 238D, 238E, 238F, 238G, 238H, 238I, 238K, 238L, 238M,238N, 238Q, 238R, 238S, 238T, 238V, 238W, 238Y, 239D, 239E, 239F, 239G,239H, 239I, 239K, 239L, 239M, 239N, 239P, 239Q, 239R, 239T, 239V, 239W,239Y, 240A, 240I, 240M, 240T, 241D, 241E, 241L, 241R, 241S, 241W, 241Y,243E, 243H, 243L, 243Q, 243R, 243W, 243Y, 244H, 245A, 246D, 246E, 246H,246Y, 247G, 247V, 249H, 249Q, 249Y, 255E, 255Y, 258H, 258S, 258Y, 260D,260E, 260H, 260Y, 262A, 262E, 262F, 262I, 262T, 263A, 263I, 263M, 263T,264A, 264D, 264E, 264F, 264G, 264H, 264I, 264K, 264L, 264M, 264N, 264P,264Q, 264R, 264S, 264T, 264W, 264Y, 265F, 265G, 265H, 265I, 265K, 265L,265M, 265N, 265P, 265Q, 265R, 265S, 265T, 265V, 265W, 265Y, 266A, 266I,266M, 266T, 267D, 267E, 267F, 267H, 267I, 267K, 267L, 267M, 267N, 267P,267Q, 267R, 267T, 267V, 267W, 267Y, 268D, 268E, 268F, 268G, 268I, 268K,268L, 268M, 268P, 268Q, 268R, 268T, 268V, 268W, 269F, 269G, 269H, 269I,269K, 269L, 269M, 269N, 269P, 269R, 269S, 269T, 269V, 269W, 269Y, 270F,270G, 270H, 270I, 270L, 270M, 270P, 270Q, 270R, 270S, 270T, 270W, 270Y,271A, 271D, 271E, 271F, 271G, 271H, 271I, 271K, 271L, 271M, 271N, 271Q,271R, 271S, 271T, 271V, 271W, 271Y, 272D, 272F, 272G, 272H, 272I, 272K,272L, 272M, 272P, 272R, 272S, 272T, 272V, 272W, 272Y, 273I, 274D, 274E,274F, 274G, 274H, 274I, 274L, 274M, 274N, 274P, 274R, 274T, 274V, 274W,274Y, 275L, 275W, 276D, 276E, 276F, 276G, 276H, 276I, 276L, 276M, 276P,276R, 276S, 276T, 276V, 276W, 276Y, 278D, 278E, 278G, 278H, 278I, 278K,278L, 278M, 278N, 278P, 278Q, 278R, 278S, 278T, 278V, 278W, 280G, 280K,280L, 280P, 280W, 281D, 281E, 281K, 281N, 281P, 281Q, 281Y, 282E, 282G,282K, 282P, 282Y, 283G, 283H, 283K, 283L, 283P, 283R, 283Y, 284D, 284E,284L, 284N, 284Q, 284T, 284Y, 285D, 285E, 285K, 285Q, 285W, 285Y, 286E,286G, 286P, 286Y, 288D, 288E, 288Y, 290D, 290H, 290L, 290N, 290W, 291D,291E, 291G, 291H, 291I, 291Q, 291T, 292D, 292E, 292T, 292Y, 293F, 293G,293H, 293I, 293L, 293M, 293N, 293P, 293R, 293S, 293T, 293V, 293W, 293Y,294F, 294G, 294H, 294I, 294K, 294L, 294M, 294P, 294R, 294S, 294T, 294V,294W, 294Y, 295D, 295E, 295F, 295G, 295H, 295I, 295M, 295N, 295P, 295R,295S, 295T, 295V, 295W, 295Y, 296A, 296D, 296E, 296G, 296H, 296I, 296K,296L, 296M, 296N, 296Q, 296R, 296S, 296T, 296V, 297D, 297E, 297F, 297G,297H, 297I, 297K, 297L, 297M, 297P, 297Q, 297R, 297S, 297T, 297V, 297W,297Y, 298A, 298D, 298E, 298F, 298H, 298I, 298K, 298M, 298N, 298Q, 298R,298T, 298W, 298Y, 299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L,299M, 299N, 299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 300A, 300D, 300E,300G, 300H, 300K, 300M, 300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W,301D, 301E, 301H, 301Y, 302I, 303D, 303E, 303Y, 304D, 304H, 304L, 304N,304T, 305E, 305T, 305Y, 313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y,320D, 320F, 320G, 320H, 320I, 320L, 320N, 320P, 320S, 320T, 320V, 320W,320Y, 322D, 322F, 322G, 322H, 322I, 322P, 322S, 322T, 322V, 322W, 322Y,323I, 324D, 324F, 324G, 324H, 324I, 324L, 324M, 324P, 324R, 324T, 324V,324W, 324Y, 325A, 325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M,325P, 325Q, 325R, 325S, 325T, 325V, 325W, 325Y, 326F, 326I, 326L, 326P,326T, 327D, 327E, 327F, 327H, 327I, 327K, 327L, 327M, 327N, 327P, 327R,327S, 327T, 327V, 327W, 327Y, 328A, 328D, 328E, 328F, 328G, 328H, 328I,328K, 328M, 328N, 328P, 328Q, 328R, 328S, 328T, 328V, 328W, 328Y, 329D,329E, 329F, 329G, 329H, 329I, 329K, 329L, 329M, 329N, 329Q, 329R, 329S,329T, 329V, 329W, 329Y, 330E, 330F, 330G, 330H, 330I, 330L, 330M, 330N,330P, 330R, 330S, 330T, 330V, 330W, 330Y, 331D, 331F, 331H, 331I, 331L,331M, 331Q, 331R, 331T, 331V, 331W, 331Y, 332A, 332D, 332E, 332F, 332H,332K, 332L, 332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, 332Y,333A, 333F, 333H, 333I, 333L, 333M, 333P, 333T, 333Y, 334A, 334F, 334I,334L, 334P, 334T, 335D, 335F, 335G, 335H, 335I, 335L, 335M, 335N, 335P,335R, 335S, 335V, 335W, 335Y, 336E, 336K, 336Y, 337E, 337H, and 337N,wherein numbering is according to the EU index.

In further aspects, the amino acid modification can be at a positionselected from the group consisting of 221, 222, 223, 224, 225, 227, 228,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 245,246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 274, 275, 276, 278, 280, 281, 281, 283, 284, 285, 286,288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 313, 317, 318, 320, 322, 324, 325, 326, 327, 328, 329,330, 331, 332, 333, 334, 335, 336, and 337. In additional aspects, thesubstitution can be selected from the group consisting of 221K, 222Y,223E, 223K, 224E, 224Y, 225E, 225W, 227E, 227G, 227K, 227Y, 228E, 228G,228K, 228Y, 230A, 230E, 230G, 230Y, 231E, 231G, 231K, 231P, 231Y, 232E,232G, 232K, 232Y, 233A, 233F, 233H, 233I, 233K, 233L, 233M, 233N, 233Q,233R, 233S, 233T, 233V, 233W, 233Y, 234D, 234E, 234F, 234G, 234H, 234I,234K, 234M, 234N, 234P, 234Q, 234R, 234S, 234T, 234W, 234Y, 235D, 235F,235G, 235H, 235I, 235K, 235M, 235N, 235Q, 235R, 235S, 235T, 235V, 235W,235Y, 236D, 236E, 236F, 236H, 236I, 236K, 236L, 236M, 236N, 236P, 236Q,236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H, 237I, 237K,237L, 237M, 237N, 237P, 237Q, 237R, 237S, 237T, 237V, 237W, 237Y, 238D,238E, 238F, 238G, 238H, 238I, 238K, 238L, 238M, 238N, 238Q, 238R, 238S,238T, 238V, 238W, 238Y, 239D, 239E, 239F, 239G, 239H, 239I, 239K, 239L,239M, 239N, 239P, 239Q, 239R, 239T, 239V, 239W, 239Y, 240M, 240T, 241D,241E, 241R, 241S, 241W, 241Y, 243E, 243H, 243Q, 243R, 243W, 243Y, 245A,246D, 246H, 246Y, 247G, 247V, 249H, 249Q, 249Y, 255E, 255Y, 258H, 258S,258Y, 260D, 260E, 260H, 260Y, 262A, 262E, 262F, 262I, 262T, 263A, 263I,263M, 263T, 264D, 264E, 264F, 264G, 264H, 264I, 264K, 264L, 264M, 264N,264P, 264Q, 264R, 264S, 264T, 264W, 264Y, 265F, 265G, 265H, 265I, 265K,265L, 265M, 265P, 265Q, 265R, 265S, 265T, 265V, 265W, 265Y, 266A, 266I,266M, 266T, 267D, 267E, 267F, 267H, 267I, 267K, 267L, 267M, 267N, 267P,267Q, 267R, 267V, 267W, 267Y, 268F, 268G, 268I, 268M, 268P, 268Q, 268V,268W, 269F, 269G, 269H, 269I, 269L, 269M, 269N, 269P, 269R, 269S, 269T,269V, 269W, 269Y, 270F, 270G, 270H, 270I, 270L, 270M, 270P, 270Q, 270R,270S, 270T, 270W, 270Y, 271A, 271D, 271E, 271F, 271G, 271H, 271I, 271K,271L, 271M, 271N, 271Q, 271R, 271S, 271T, 271V, 271W, 271Y, 272F, 272G,272H, 272I, 272K, 272L, 272M, 272P, 272R, 272S, 272T, 272V, 272W, 272Y,274D, 274E, 274F, 274G, 274H, 274I, 274L, 274M, 274P, 274R, 274T, 274V,274W, 274Y, 275W, 276D, 276E, 276F, 276G, 276H, 276I, 276L, 276M, 276P,276R, 276S, 276T, 276V, 276W, 278D, 278E, 278G, 278H, 278I, 278K, 278L,278M, 278N, 278P, 278Q, 278R, 278S, 278T, 278V, 278W, 280G, 280P, 280W,281E, 281K, 281N, 281P, 281Y, 282G, 282P, 282Y, 283G, 283H, 283K, 283L,283P, 283R, 283Y, 284L, 284N, 284Q, 284T, 284Y, 285K, 285Q, 285W, 285Y,286G, 286P, 286Y, 288Y, 290H, 290L, 290W, 291D, 291E, 291G, 291H, 291I,291Q, 291T, 292D, 292E, 292T, 292Y, 293F, 293G, 293H, 293I, 293L, 293M,293N, 293P, 293R, 293S, 293T, 293W, 293Y, 294F, 294G, 294H, 294I, 294K,294L, 294M, 294P, 294R, 294S, 294T, 294V, 294W, 294Y, 295D, 295F, 295G,295H, 295I, 295M, 295N, 295P, 295R, 295S, 295T, 295V, 295W, 295Y, 296A,296D, 296E, 296G, 296I, 296K, 296L, 296M, 296N, 296Q, 296R, 296S, 296T,296V, 297D, 297E, 297F, 297G, 297H, 297I, 297K, 297L, 297M, 297P, 297R,297S, 297T, 297V, 297W, 297Y, 298E, 298F, 298H, 298I, 298K, 298M, 298Q,298R, 298W, 298Y, 299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L,299M, 299N, 299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 300A, 300D, 300E,300G, 300H, 300K, 300M, 300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W,301D, 301E, 301Y, 302I, 303D, 303E, 303Y, 304H, 304L, 304N, 304T, 305E,305T, 305Y, 313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y, 320D, 320F,320G, 320H, 320I, 320L, 320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D,322F, 322G, 322H, 322I, 322P, 322S, 322T, 322V, 322W, 322Y, 324D, 324F,324G, 324H, 324I, 324L, 324M, 324P, 324R, 324T, 324V, 324W, 324Y, 325A,325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q, 325R,325S, 325T, 325V, 325W, 325Y, 326L, 326P, 326T, 327D, 327E, 327F, 327H,327I, 327K, 327L, 327M, 327P, 327R, 327V, 327W, 327Y, 328A, 328D, 328E,328F, 328G, 328H, 328K, 328M, 328N, 328P, 328Q, 328R, 328S, 328T, 328V,328W, 328Y, 329D, 329E, 329F, 329G, 329H, 329I, 329K, 329L, 329M, 329N,329Q, 329R, 329S, 329T, 329V, 329W, 329Y, 330E, 330F, 330H, 330I, 330L,330M, 330N, 330P, 330W, 330Y, 331D, 331F, 331H, 331I, 331L, 331M, 331Q,331R, 331T, 331V, 331W, 331Y, 332A, 332F, 332H, 332L, 332M, 332N, 332P,332Q, 332R, 332T, 332V, 332W, 332Y, 333F, 333H, 333I, 333L, 333M, 333P,333T, 333Y, 334F, 334P, 334T, 335D, 335F, 335G, 335H, 335I, 335L, 335M,335P, 335R, 335S, 335V, 335W, 335Y, 336E, 336K, 336Y, 337H, and 337N.

In further aspect, the modification is at a position selected from thegroup consisting of 221, 222, 223, 224, 225, 228, 230, 231, 232, 240,244, 245, 247, 262, 263, 266, 271, 273, 275, 281, 284, 291, 299, 302,304, 313, 323, 325, 328, 332, 336, wherein the positional numbering isaccording to the EU index. In additional aspects, the modification isselected from the group consisting of 221K, 221Y, 222E, 222Y, 223E,223K, 224E, 224Y, 225E, 225K, 225W, 228E, 228G, 228K, 228Y, 230A, 230E,230G, 230Y, 231E, 231G, 231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 240A,240I, 240M, 240T, 244H, 245A, 247G, 247V, 262A, 262E, 262F, 262I, 262T,263A, 263I, 263M, 263T, 266A, 266I, 266M, 266T, 271A, 271D, 271E, 271F,271G, 271H, 271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S, 271T, 271V,271W, 271Y, 273I, 275L, 275W, 281D, 281E, 281K, 281N, 281P, 281Q, 281Y,284D, 284E, 284L, 284N, 284Q, 284T, 284Y, 291D, 291E, 291G, 291H, 291I,291Q, 291T, 299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M,299N, 299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 304D, 304H, 304L, 304N,304T, 313F, 323I, 325A, 325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L,325M, 325P, 325Q, 325R, 325S, 325T, 325V, 325W, 325Y, 328A, 328D, 328E,328F, 328G, 328H, 328I, 328K, 328M, 328N, 328P, 328Q, 328R, 328S, 328T,328V, 328W, 328Y, 332A, 332D, 332E, 332F, 332H, 332K, 332L, 332M, 332N,332P, 332Q, 332R, 332S, 332T, 332V, 332W, 332Y, 336E, 336K, and 336Y.

The antibody can further include a second amino acid modification at aposition selected from the group consisting of 221, 222, 223, 224, 225,227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 281,283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 313, 317, 318, 320, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and 337,wherein numbering is according to the EU index. The amino acidmodification can be a substitution selected from the group consisting of221K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E,227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231E,231G, 231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G,233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W,233Y, 234A, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234P,234Q, 234R, 234S, 234T, 234V, 234W, 234Y, 235A, 235D, 235E, 235F, 235G,235H, 235I, 235K, 235M, 235N, 235P, 235Q, 235R, 235S, 235T, 235V, 235W,235Y, 236A, 236D, 236E, 236F, 236H, 236I, 236K, 236L, 236M, 236N, 236P,236Q, 236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H, 237I,237K, 237L, 237M, 237N, 237P, 237Q, 237R, 237S, 237T, 237V, 237W, 237Y,238D, 238E, 238F, 238G, 238H, 238I, 238K, 238L, 238M, 238N, 238Q, 238R,238S, 238T, 238V, 238W, 238Y, 239D, 239E, 239F, 239G, 239H, 239I, 239K,239L, 239M, 239N, 239P, 239Q, 239R, 239T, 239V, 239W, 239Y, 240A, 240I,240M, 240T, 241D, 241E, 241L, 241R, 241S, 241W, 241Y, 243E, 243H, 243L,243Q, 243R, 243W, 243Y, 244H, 245A, 246D, 246E, 246H, 246Y, 247G, 247V,249H, 249Q, 249Y, 255E, 255Y, 258H, 258S, 258Y, 260D, 260E, 260H, 260Y,262A, 262E, 262F, 262I, 262T, 263A, 263I, 263M, 263T, 264A, 264D, 264E,264F, 264G, 264H, 264I, 264K, 264L, 264M, 264N, 264P, 264Q, 264R, 264S,264T, 264W, 264Y, 265F, 265G, 265H, 265I, 265K, 265L, 265M, 265N, 265P,265Q, 265R, 265S, 265T, 265V, 265W, 265Y, 266A, 266I, 266M, 266T, 267D,267E, 267F, 267H, 267I, 267K, 267L, 267M, 267N, 267P, 267Q, 267R, 267T,267V, 267W, 267Y, 268D, 268E, 268F, 268G, 268I, 268K, 268L, 268M, 268P,268Q, 268R, 268T, 268V, 268W, 269F, 269G, 269H, 269I, 269K, 269L, 269M,269N, 269P, 269R, 269S, 269T, 269V, 269W, 269Y, 270F, 270G, 270H, 270I,270L, 270M, 270P, 270Q, 270R, 270S, 270T, 270W, 270Y, 271A, 271D, 271E,271F, 271G, 271H, 271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S, 271T,271V, 271W, 271Y, 272D, 272F, 272G, 272H, 272I, 272K, 272L, 272M, 272P,272R, 272S, 272T, 272V, 272W, 272Y, 273I, 274D, 274E, 274F, 274G, 274H,274I, 274L, 274M, 274N, 274P, 274R, 274T, 274V, 274W, 274Y, 275L, 275W,276D, 276E, 276F, 276G, 276H, 276I, 276L, 276M, 276P, 276R, 276S, 276T,276V, 276W, 276Y, 278D, 278E, 278G, 278H, 278I, 278K, 278L, 278M, 278N,278P, 278Q, 278R, 278S, 278T, 278V, 278W, 280G, 280K, 280L, 280P, 280W,281D, 281E, 281K, 281N, 281P, 281Q, 281Y, 282E, 282G, 282K, 282P, 282Y,283G, 283H, 283K, 283L, 283P, 283R, 283Y, 284D, 284E, 284L, 284N, 284Q,284T, 284Y, 285D, 285E, 285K, 285Q, 285W, 285Y, 286E, 286G, 286P, 286Y,288D, 288E, 288Y, 290D, 290H, 290L, 290N, 290W, 291D, 291E, 291G, 291H,291I, 291Q, 291T, 292D, 292E, 292T, 292Y, 293F, 293G, 293H, 293I, 293L,293M, 293N, 293P, 293R, 293S, 293T, 293V, 293W, 293Y, 294F, 294G, 294H,294I, 294K, 294L, 294M, 294P, 294R, 294S, 294T, 294V, 294W, 294Y, 295D,295E, 295F, 295G, 295H, 295I, 295M, 295N, 295P, 295R, 295S, 295T, 295V,295W, 295Y, 296A, 296D, 296E, 296G, 296H, 296I, 296K, 296L, 296M, 296N,296Q, 296R, 296S, 296T, 296V, 297D, 297E, 297F, 297G, 297H, 297I, 297K,297L, 297M, 297P, 297Q, 297R, 297S, 297T, 297V, 297W, 297Y, 298A, 298D,298E, 298F, 298H, 298I, 298K, 298M, 298N, 298Q, 298R, 298T, 298W, 298Y,299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M, 299N, 299P,299Q, 299R, 299S, 299V, 299W, 299Y, 300A, 300D, 300E, 300G, 300H, 300K,300M, 300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W, 301D, 301E, 301H,301Y, 302I, 303D, 303E, 303Y, 304D, 304H, 304L, 304N, 304T, 305E, 305T,305Y, 313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y, 320D, 320F, 320G,320H, 320I, 320L, 320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D, 322F,322G, 322H, 322I, 322P, 322S, 322T, 322V, 322W, 322Y, 323I, 324D, 324F,324G, 324H, 324I, 324L, 324M, 324P, 324R, 324T, 324V, 324W, 324Y, 325A,325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q, 325R,325S, 325T, 325V, 325W, 325Y, 326E, 326I, 326L, 326P, 326T, 327D, 327E,327F, 327H, 327I, 327K, 327L, 327M, 327N, 327P, 327R, 327S, 327T, 327V,327W, 327Y, 328A, 328D, 328E, 328F, 328G, 328H, 328I, 328K, 328M, 328N,328P, 328Q, 328R, 328S, 328T, 328V, 328W, 328Y, 329D, 329E, 329F, 329G,329H, 329I, 329K, 329L, 329M, 329N, 329Q, 329R, 329S, 329T, 329V, 329W,329Y, 330E, 330F, 330G, 330H, 330I, 330L, 330M, 330N, 330P, 330R, 330S,330T, 330V, 330W, 330Y, 331D, 331F, 331H, 331I, 331L, 331M, 331Q, 331R,331T, 331V, 331W, 331Y, 332A, 332D, 332E, 332F, 332H, 332K, 332L, 332M,332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, 332Y, 333A, 333F, 333H,333I, 333L, 333M, 333P, 333T, 333Y, 334A, 334F, 334I, 334L, 334P, 334T,335D, 335F, 335G, 335H, 335I, 335L, 335M, 335N, 335P, 335R, 335S, 335V,335W, 335Y, 336E, 336K, 336Y, 337E, 337H, and 337N, wherein numbering isaccording to the EU index.

In further aspects, the amino acid modification is 332E. The secondamino acid modification can be selected from the group consisting of:236A, 239D, 332E, 268D, 268E, 330Y, and 330L. In certain preferredembodiments, the second amino acid modification is 239D.

In other aspects, the modification is a glycoform modification thatreduces the level of fucose relative to the parent antibody. In stillother aspects, the invention is directed to a composition includingplurality of glycosylated antibodies, wherein about 80-100% of theglycosylated antibodies in the composition comprise a mature corecarbohydrate structure which lacks fucose.

In a further embodiment, the antibody reduces binding to FcγRIIb ascompared to the parent anti-CD19 antibody.

In another aspect, the invention is directed to an antibody that bindsCD19 and includes a heavy chain and/or a light chain. The heavy chainhas a CDR1 comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO: 137 and 138, a CDR2 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:111-115 andCDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 116-118. The light chain has a CDR1 comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs:119-128, a CDR2 comprising the amino acid sequence of SEQ ID NOs:129,and a CDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:130-131.

In further variations, the antibody has a variable heavy chain sequenceselected from the group consisting of SEQ ID NOS: 13-16, 20-23, and27-44, and/or a variable light chain sequence selected from the groupconsisting of SEQ ID NOS: 17-19, 24-26, and 45-79.

In still further variations, the antibody includes a heavy chainsequence selected from the group consisting of SEQ ID NOS: 86-95, and/ora light chain sequence selected from the group consisting of SEQ ID NOS:96-110.

In various additional aspects, the invention is directed to a nucleicacid sequence encoding any of the antibodies disclosed herein.

In further aspects, the invention is directed to a method of treating aB-cell related disease by administering an antibody according to claim1. In certain variations, the disease is selected from non-Hodgkin'slymphomas (NHL), chronic lymphocytic leukemia (CLL), B-cell acutelymphoblastic leukemia/lymphoma (B-ALL), and mantle cell lymphoma (MCL).In certain aspects, the disease is an autoimmune disease, such asrheumatoid arthritis (RA), systemic lupus erythematosus (SLE or lupus),multiple sclerosis, Sjogren's syndrome, and idiopathic thrombocytopeniapurpura (ITP).

In further aspects, the invention is directed to a compositioncomprising an antibody described herein and an acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings further illustrate aspects of the invention, andare not meant to constrain the invention to any particular applicationor theory of operation.

FIG. 1: Amino acid sequence of homo sapiens CD19, as obtained from cDNAclone MGC:12802, IMAGE:4504919, GenBank Accession:BC006338.

FIG. 2. Sequences of the natural antibody constant regions, includingthe kappa constant light chain, and the gamma constant heavy chains forIgG1, IgG2, IgG3, and IgG4. Also provided is the sequence of a HybridIgG constant chain, and a Hybrid IgG constant chain comprising thesubstitutions 239D and I332E.

FIGS. 3A and 3B. Alignment of the amino acid sequences of the human IgGimmunoglobulins IgG1, IgG2, IgG3, and IgG4. FIG. 3A provides thesequences of the CH1 (Cγ1) and hinge domains, and FIG. 3B provides thesequences of the CH2 (Cγ2) and CH3 (Cγ3) domains. Positions are numberedaccording to the EU index of the IgG1 sequence, and differences betweenIgG1 and the other immunoglobulins IgG2, IgG3, and IgG4 are shown ingray. Allotypic polymorphisms exist at a number of positions, and thusslight differences between the presented sequences and sequences in theprior art may exist. The possible beginnings of the Fc region arelabeled, defined herein as either EU position 226 or 230.

FIGS. 4A and 4B. The common haplotypes of the gamma chain of human IgG1(FIG. 4A) and IgG2 (FIG. 4B) showing the positions and the relevantamino acid substitutions.

FIG. 5. Preferred embodiments of receptor binding profiles that includeincreases to, reductions to, or no effect to the binding to variousreceptors, where such changes may be beneficial in certain contexts.

FIGS. 6A and 6B. Amino acid sequences of the heavy chain and light chainvariable regions of the original 4G7 and HD37 antibodies (H0 and L0).FIG. 6A provides the sequences of the VH and VL domains, and FIG. 6Bprovides the sequences of the CDRs. CDR boundaries are determinedaccording to the convention of Kabat (VH CDR1: 31-35b, VH CDR2: 50-65,VH CDR3: 95-102, VL CDR1: 24-34, VL CDR2: 50-56, and VL CDR3: 89-97).

FIG. 7. The relative binding affinities of 4G7 Hybrid S239D/I332E and4G7 IgG1 antibody to a panel of Fc receptors.

FIGS. 8A and 8B. ADCC of 4G7 Hybrid S239D/I332E, HD37 HybridS239D/I332E, 4G7 IgG1, HD37 IgG1, and a negative control antibody on theDaudi cell line (FIG. 8A) and ADCC of 4G7 Hybrid S239D/I332E, 4G7 IgG1,rituximab, and a negative control antibody on the SUP-B15 and Raji celllines (FIG. 8B).

FIG. 9. A cell-surface binding assay of 4G7 Hybrid S239D/I332E to Rajicells.

FIGS. 10A and 10B. FIG. 10A shows ADCC assays of 4G7 Hybrid S239D/I332E,4G7 IgG1, and rituximab on a panel of 14 cell lines representing variouslymphomas and leukemias. Both parameters potency (EC50) and efficacy (%ADCC) are normalized to that of rituximab (anti-CD20). FIG. 10B liststested lymphoma and leukemia cell lines.

FIG. 11. Heavy chain variable region sequences with reducedimmunogenicity for anti-CD19 antibody 4G7.

FIG. 12. Light chain variable region sequences with reducedimmunogenicity for anti-CD19 antibody 4G7.

FIG. 13. Heavy chain variable region sequences with reducedimmunogenicity for anti-CD19 antibody HD37.

FIG. 14. Heavy chain variable region sequences with reducedimmunogenicity for anti-CD19 antibody HD37.

FIGS. 15, and 15B. Results of a cell-surface binding assay of reducedimmunogenicity 4G7 variants to Raji cells (FIG. 15A) and ADCC ofHD37_H2L1 Hybrid S239D/I332E and 4G7_H1L3 Hybrid S239D/I332E on MEC-1cells (FIG. 15B).

FIG. 16. Cell-binding affinity on RS4;11 cells of affinity matured 4G7relative to the H1L1 mAb.

FIG. 17. Cell-binding data to RS4;11 cells of 4G7 variants incubated for5 days at 37° C., pH 9.0 in 200 mM Tris-HCL showing the improvement instability obtained.

FIG. 18. Sequences for heavy chain variants of anti-CD19 that increaseaffinity and/or stability.

FIG. 19. Sequences for light chain variants of anti-CD19 that increaseaffinity and/or stability.

FIG. 20. Anti-proliferative properties of 4G7 Hybrid S239D/I332E on Rajicells.

FIG. 21. Anti-proliferative properties of 4G7 stability and affinityimproved Hybrid S239D/I332E on SU-DHL-6 cells with and withoutcross-linking.

FIG. 22. Phagocytosis of Raji and RS4;11 cells with 4G7 stability andaffinity improved Hybrid S239D/I332E.

FIG. 23. ADCC of 4G7 stability and affinity improved Hybrid S239D/I332Eagainst multiple lymphoma cell lines using purified natural killer (NK)cells.

FIG. 24. 4G7 stability and affinity improved Hybrid S239E/I332E bindingto 293T cells transfected with human CD19.

FIGS. 25A, 25B, and 25C. Cross-reactivity of 4G7 stability and affinityimproved Hybrid S239D/I332E to both cynomolgus and rhesus CD19.

FIG. 26. ADCC on RS4;11 and MEC-1 cells using an enhanced effectorfunction anti-CD19 antibody (4G7 H1L1 Hybrid S239D/I332E) with lowerfucose content afforded by expression in the Lec13 system.

FIG. 27. Single substitutions made for enhanced stability and/oraffinity. Variable region numbering is according to Kabat. An expandedset of positions is included in the CDRs. The canonical CDR boundariesdefined by Kabat, as listed in FIG. 6, are highlighted in gray.

FIG. 28. Anti-CD19 variable region variants constructed to optimizeaffinity and stability.

FIG. 29. Preferred variants and relative increase in binding affinityversus the parent H1L1 mAb.

FIGS. 30A and 30B. B cell proliferation assay, showing capacity ofvariant anti-CD19 antibodies to inhibit viability of primary B cells.FIG. 30A shows the dose-dependence of anti-mu antibody on B cellproliferation. FIG. 30B shows B cell proliferation in the presence offixed anti-mu (2 mg/ml) plus varying concentrations of anti-CD19 WT andFc variant, and anti-CD30 Fc variant control antibodies.Anti-Anti-CD19_IgG1_WT=4G7_H3_L1 IgG1_WT,Anti-CD19_Hybrid_S239D/I332E=4G7_H3_L1_Hybrid_239D/332E, andAnti-CD30_S239D/I332E, used here as a negative control,=AC10_H3.69V2_L3.71_Hybrid_239D/332E (as disclosed in U.S. Ser. No.11/686,853, Lazar G. A. et al., filed Mar. 15, 2007).

DETAILED DESCRIPTION OF THE INVENTION

The disclosure is directed to modified anti-CD19 antibodies and methodsof using the same. In various aspects, the antibodies can have a havinga modified Fc region, specific CDR sequences, variable region sequences,and/or constant region modifications. In various embodiments, theantibodies are humanized. The disclosure is further directed to methodsof using the antibodies in various disease indications, including thoseof B-cell origin such as B-cell origin non-Hodgkin's lymphoma (NHL),acute lymphoblastic leukemia (ALL), and autoimmune related disease.

In order that the invention may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. In various aspects, theenhanced ADCC effector function can mean enhanced potency or enhancedefficacy. By “potency” as used in the experimental context is meant theconcentration of antibody when a particular therapeutic effect isobserved EC50 (half maximal effective concentration). By “efficacy” asused in the experimental context is meant the maximal possible effectorfunction at saturating levels of antibody.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “amino acid” and “amino acid identify” as used herein is meant one ofthe 20 naturally occurring amino acids or any non-natural analogues thatmay be present at a specific, defined position. Thus “amino acid” asused herein means both naturally occurring and synthetic amino acids.For example, homophenylalanine, citrulline and noreleucine areconsidered amino acids for the purposes of the invention. “Amino acid”also includes imino acid residues such as proline and hydroxyproline.The side chain may be in either the (R) or the (S) configuration. In thepreferred embodiment, the amino acids are in the (S) or L-configuration.If non-naturally occurring side chains are used, non-amino acidsubstituents may be used, for example to prevent or retard in vivodegradation.

By “B cell” or “B lymphocyte” as used herein is meant a type oflymphocyte developed in bone marrow that circulates in the blood andlymph, and provides humoral immunity. B cells recognize free antigenmolecules and differentiate or mature into plasma cells that secreteimmunoglobulin (antibodies) that inactivate the antigens. Memory cellsare also generated that make the specific immunoglobulin (antibody) onsubsequent encounters with such antigen. B cells are also known as “Betacells” in the islet of Langerhans.

By “B-cell antigen” or “B-cell marker” as used herein is meant anyprotein that is expressed on B cells.

By “CD19” as used herein is meant the protein of SEQ ID NO:1 (depictedin FIG. 1). CD19 is also known as B-cell surface antigen B4, B-cellantigen CD19, CD19 antigen, and Leu-12. Human CD19 is designatedGeneID:930 by Entrez Gene, and HGNC:1633 by HGNC. CD19 can be encoded bythe gene designated CD19. The use of “CD19” herein is meant to encompassall known or as yet undiscovered alleles and polymorphic forms of CD19.

By “CDC” or “complement dependent cytotoxicity” as used herein is meantthe reaction wherein one or more complement protein components recognizebound antibody on a target cell and subsequently cause lysis of thetarget cell.

By “constant region” as used herein is meant the polypeptide includingat least a portion of the first three constant regions of an antibody,having at least one effector function. Thus constant region thus refersto the last three constant region immunoglobulin domains of IgA, IgD,and IgG, and the last four constant region immunoglobulin domains of IgEand IgM, and the flexible hinge N-terminal to these domains. For IgA andIgM, Fc may include the J chain. For IgG, the constant region includeimmunoglobulin domains Cgamma 1, Cgamma2 and Cgamma3 (Cγ1, Cγ2 and Cγ3)and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although theboundaries of the constant region may vary, the human IgG heavy chain Fcregion is usually defined to comprise residues to its carboxyl-terminus,wherein the numbering is according to the EU index as in Kabat. Theconstant light chain typically comprises a single domain, and as definedherein refers to positions 108-214 of Cκ or Cλ, wherein numbering isaccording to the EU index. For full length IgG antibodies, the constantheavy chain, as defined herein, refers to the N-terminus of the CH1domain to the C-terminus of the CH3 domain, or positions 118-447,wherein numbering is according to the EU index. “Constant region” mayrefer to this region in isolation, or a truncation or fusion includeantibodies, Fc fusions, isolated Fcs, and Fc fragments. In variousembodiments, the constant region may be the region of the antibody thatis encoded by one of the light or heavy chain immunoglobulin constantregion genes, i.e. the region of an antibody encoded by the kappa (Cκ)or lambda (Cλ) light chains. In various embodiments, the constant heavychain or heavy chain constant region can be the region of an antibodyencoded by the mu, delta, gamma, alpha, or epsilon genes to define theantibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include FcγR-mediated effectorfunctions such as ADCC and ADCP, and complement-mediated effectorfunctions such as CDC. By “effector cell” as used herein is meant a cellof the immature system that expresses one or more Fc receptors andmediates one or more effector functions. Effector cells include but arenot limited to monocytes, macrophages, neutrophils, dendritic cells,eosinophils, mast cells, platelets, B cells, large granular lymphocytes,Langerhans' cells, natural killer (NK) cells, and γδ T cells, and may befrom any organism including but not limited to humans, mice, rats,rabbits, and monkeys.

By “Fab” or “Fab region” as used herein is meant the polypeptides thatcomprise the V_(H), CH1, V_(H), and C_(L) immunoglobulin domains. Fabmay refer to this region in isolation, or this region in the context ofa fully length antibody or antibody fragment.

By “Fc” or “Fc region”, as used herein is meant the polypeptidecomprising the constant region of no antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domainsCgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1)and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to compriseresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as in Kabat. Fc may refer to this region inisolation, or this region in the context of an Fc polypeptide, forexample an antibody. By “Fc polypeptide” as used herein is meantpolypeptide that comprises all or part of an Fc region. Fc polypeptideinclude antibodies, Fc fusions, isolated Fcs, and Fc fragments.

By “Fc gamma receptor” or “FcγR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region. In variousembodiments, FcγR are substantially encoded by the FcγR genes. In humansthis family includes but is not limited to FcγRI (CD64), includingisoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoformsFcγRIIa (including allotypes H131 and R131), FcγRIIb (includingFcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), includingisoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb(including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA1) (Jefferis et al.,2002, Immunol Lett 82:57-65, incorporated entirely by reference), aswell as any undiscovered human FcγRs or FcγR isoforms or allotypes.Mouse FcγRa include but are not limited to FcγRI (CD64), FcγRII (CD32),FcRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouseFcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism,including but not limited to humans, mice, rats, rabbits, and monkeys.

By “Fc ligand” or “Fc receptor” as used herein is meant a molecule,preferably a polypeptide, from any organism that binds to the Fc regionof an antibody to form an Fc-ligand complex. Fc ligands include but arenot limited to FcγRs, FcRn, C1q, C3, mannan binding lectin, mannosereceptor, staphylococcal protein A, streptococcal protein G, and viralFcγR. Fc ligands also include Fc receptor homologs (FcRH), which are afamily of Fc receptors that are homologous to the FcγRs (Davis et al.,2002, Immunological Reviews 190:123-136, incorporated entirely byreference). Fc ligands may include undiscovered molecules that bind Fc.

By “IgG” as used herein is meant a polypeptide belonging to the class ofantibodies that are substantially encoded by a recognized immunoglobulingamma gene. In humans this class comprises IgG1, IgG2, IgG3, and IgG4.In mice this class comprises IgG1, IgG2a, IgG2b, IgG3. By“immunoglobulin (Ig)” herein is meant a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes.Immunoglobulins include tub are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full length antibodies, antibody fragments, and individualimmunoglobulin domains. By “immunoglobulin (Ig) domain” herein is meanta region of an immunoglobulin that exists as a distinct structuralentity as ascertained by one skilled in the art of protein structure. Igdomains typically have a characteristic β-sandwich folding topology. Theknown Ig domains in the IgG class of antibodies are V_(H), Cγ1, Cγ2,Cγ3, V_(L), and C_(L).

By “modification” herein is meant an alteration in the physical,chemical, or sequence properties of a protein, polypeptide, antibody, orimmunoglobulin. Preferred modifications of the invention are amino acidmodifications and glycoform modifications.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. By “amino acidsubstitution” or “substitution” herein is meant the replacement of anamino acid at a particular position in a parent polypeptide sequencewith another amino acid. For example, the substitution I332E refers to avariant polypeptide, in this case a constant heavy chain variant, inwhich the isoleucine at position 332 is replaced with glutamic acid. TheWT residue may or may not be designated. For the preceding example, 332Eindicates the substitution of position 332 with a glutamic acid. For thepurposes herein, multiple substitutions are typically separated by aslash. For example, 239D/332E refers to a double variant comprising thesubstitution 239D and 332E. By “amino acid insertion” or “insertion” asused herein is meant the addition of an amino acid at a particularposition in a parent polypeptide sequence. For example, insert -236designates an insertion of glycine at position 236. By “amino aciddeletion” or “deletion” as used herein is meant the removal of an aminoacid at a particular position in a parent polypeptide sequence. Forexample, G236-designates the deletion of glycine at position 236.

By “glycoform modification” or “modified glycoform” or “engineeredglycoform” as used herein is meant a carbohydrate composition that iscovalently attached to a protein, for example an antibody, wherein saidcarbohydrate composition differs chemically from that of a parentprotein. Modified glycoform typically refers to the differentcarbohydrate or oligosaccharide; thus for example an antibody maycomprise a modified glycoform. Alternatively, modified glycoform mayrefer to the antibody that comprises the different carbohydrate oroligosaccharide.

By “parent polypeptide”, “parent protein”, “precursor polypeptide”, or“precursor protein” as used herein is meant an unmodified polypeptidethat is subsequently modified to generate a variant. Said parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent antibody” or “parent immunoglobulin” as usedherein is meant an antibody or immunoglobulin that is modified togenerate a variant. By “parent anti-CD19 antibody” or “parent anti-CD19immunoglobulin” as used herein is meant an antibody or immunoglobulinthat binds CD19 and is modified to generate a variant.

By “protein” or “polypeptide” as used herein is meant at least twocovalently attached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The protein may be made up of naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index as in Kabat. Correspondingpositions are determined as outlined herein, generally through alignmentwith other parent sequences.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 and N297) is a residue at position 297 in thehuman antibody IgG1.

By “target antigen” or “target” or “antigen” as used herein is meant themolecule that is bound specifically by the variable region of a givenantibody. A target antigen may be a protein, carbohydrate, lipid, orother chemical compound. By “target cell” as used herein is meant a cellthat expresses a target antigen.

By “variable region” is meant the variable region of an antibody heavychain or light chain. The heavy chain variable region (VH), as definedherein, refers to the N-terminus to the C-terminus of the VH domain,defined by residues 1-113 according to the numbering convention ofKabat. The light chain variable region (VL), as defined herein, refersto the N-terminus to the C-terminus of the VL domain, defined byresidues 1-107 according to the numbering convention of Kabat. Thoseskilled in the art will recognize that the Kabat variable regionnumbering convention employs letters to account for the variable lengthof CDRs. Thus that a VH is defined by Kabat residues 1-113, and that aVL is defined by Kabat 1-107, does not necessarily mean that the VHdomain contains exactly 113 residues, nor that VL contains exactly 107residues. Rather, residues 1-113 of VH and 1-107 of VL according toKabat are meant to encompass the structural domains that were determinedby sequence alignments of a large set of variable length antibodyvariable regions of varying length ((Kabat et al., 1991, Sequences ofProteins of Immunological Interest, 5th Ed., United States Public HealthService, National Institutes of Health, Bethesda, incorporated entirelyby reference). In certain embodiments, the variable region can comprisesone or more Ig domains substantially encoded by any of the Vκ, Vλ,and/or V_(H) gene that makes up the kappa, lambda, and heavy chainimmunoglobulin genetic loci respectively.

By “variant protein”, “protein variant”, “variant polypeptide”, or“polypeptide variant” as used herein is meant a polypeptide sequencethat differs from that of a parent polyeptide sequence by virtue of atleast one amino acid modification. Variant polypeptide may refer to thepolypeptide itself, a composition comprising the polypeptide, or theamino sequence that encodes it. Preferably, the variant polypeptide hasat least one amino acid modification compared to the parent polypeptide,e.g. from about one to about ten amino acid modifications, andpreferably from about one to about five amino acid modificationscompared to the parent. The variant polypeptide sequence herein willpreferably possess at least about 80% homology with a parent polypeptidesequence, and most preferably at least about 90% homology, morepreferably at least about 95% homology. Accordingly, by “variantantibody” or “antibody variant” as used herein is meant an antibodysequence that differs from that of a parent antibody sequence by virtueof at least one amino acid modification. Antibody variant may refer tothe antibody polypeptide itself, compositions comprising the antibodyvariant polypeptide, or the amino acid sequence that encodes it.Accordingly, by “variant antibody” or “antibody variant” as used hereinis meant an antibody, as defined above, that differs in sequence fromthat of a parent antibody sequence by virtue of at least one amino acidmodification. Variant antibody may refer to the protein itself,compositions comprising the protein, or the amino acid sequence thatencode it. Accordingly, by “constant heavy chain variant” or “constantlight chain variant” or “Fc variant” as used herein is meant a constantheavy chain, constant light chain, or Fc region polypeptide or sequence,respectively, that differs in sequence from that of a parent sequence byvirtue of at least one amino acid modification.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein, polypeptide, antibody, immunoglobulin, IgG,etc., has an amino acid sequence or a nucleotide sequence that has notbeen intentionally modified.

For all immunoglobulin heavy chain constant region positions discussedin the present invention, numbering is according to the EU index as inKabat (Kabat et al., 1991, Sequences of Proteins of ImmunologicalInterest, 5th Ed., United States Public Health Service, NationalInstitutes of Health, Bethesda, incorporated entirely by reference). The“EU index as in Kabat” refers to the residue numbering of the human IgG1EU antibody, as described in Edelman et al., 1969, Biochemistry63:78-85, incorporated entirely by reference.

Antibodies

As used herein, the term “antibody” refers to a monomeric or multimericprotein comprising one or more polypeptide chains. An antibody bindsspecifically to an antigen (e.g. CD19) and may be able to modulate thebiological activity of the antigen. As used herein, the term “antibody”can include “full length antibody” and “Fc polypeptide.”

By “full length antibody” herein is meant the structure that constitutesthe natural biological form of an antibody, including variable andconstant regions. For example, in most mammals, including humans andmice, the full length antibody of the IgG class is a tetramer andconsists of two identical pairs of two immunoglobulin domains V_(L) andC_(L), and each heavy chain comprising immunoglobulin domains V_(H), CH1(Cγ1), CH2 (Cγ2), and CH3 (Cγ3). In some mammals, for example in camelsand llamas, IgG antibodies may consist of only two heavy chains, eachheavy chain comprising a variable domain attached to the Fc region.

The term “antibody” also includes antibody fragments. Specific antibodyfragments include, but are not limited to, (i) the Fab fragmentconsisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward et al., 1989, Nature 341:544-546) which consists of a singlevariable, (v) isolated CDR regions, (vi) F(ab′)2 fragments a bivalentfragment comprising two linked Fab fragments (vii) single chain Fvmolecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al., 1988, Science 242:423-426, Huston etal., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883), (viii)bispecific single chain Fv dimers (PCT/US92/09965) and (ix) “diabodies”or “triabodies”, multivalent or multispecific fragments constructed bygene fusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479;WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A.90:6444-6448). In certain embodiments, antibodies are produced byrecombinant DNA techniques. Other examples of antibody formats andarchitectures are described in Holliger & Hudson, 2006, NatureBiotechnology 23(9):1126-1136, and Carter 2006, Nature ReviewsImmunology 6:343-357 and references cited therein, all expresslyincorporated by reference. In additional embodiments, antibodies areproduced by enzymatic or chemical cleavage of naturally occurringantibodies.

Natural antibody structural units typically comprise a tetramer. Eachtetramer is typically composed of two identical pairs of polypeptidechains, each pair having one “light” (typically having a molecularweight of about 25 kDa) and one “heavy” chain (typically having amolecular weight of about 50-70 kDa). Each of the light and heavy chainsare made up of two distinct regions, referred to as the variable andconstant regions. For the IgG class of immunoglobulins, the heavy chainis composed of four immunoglobulin domains linked from N- to C-terminusin the order V_(H)-CH1-CH2-CH3, referring to the heavy chain variabledomain, heavy chain constant domain 1, heavy chain constant domain 2,and heavy chain constant domain 3 respectively (also referred to asV_(H)-Cγ1-Cγ2-Cγ3, referring to the heavy chain constant domain,constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3domain respectively). The IgG light chain is composed of twoimmunoglobulin domains linked from N- to C-terminus in the orderV_(L)-C_(L), referring to the light chain variable domain and the lightchain constant domain respectively. The constant regions show lesssequence diversity, and are responsible for binding a number of naturalproteins to elicit important biochemical events.

The variable region of an antibody contains the antigen bindingdeterminants of the molecule, and thus determines the specificity of anantibody for its target antigen. The variable region is so named becauseit is the most distinct in sequence from other antibodies within thesame class. In the variable region, three loops are gathered for each ofthe V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.There are 6 CDRs total, three each per heavy and light chain, designatedV_(H) CDR1, V_(H) CDR2, V_(H) CDR3, V_(L) CDR1, V_(L) CDR2, and V_(L)CDR3. The variable region outside of the CDRs is referred to as theframework (FR) region. Although not as diverse as the CDRs, sequencevariability does occur in the Fr region between different antibodies.Overall, this characteristic architecture of antibodies provides astable scaffold (the FR region) upon which substantial antigen bindingdiversity (the CDRs) can be explored by the immune system to obtainspecificity for a broad array of antigens. A number of high-resolutionstructures are available for a variety of variable region fragments fromdifferent organisms, some unbound and some in complex with antigen.Sequence and structural features of antibody variable regions aredisclosed, for example, in Morea et al., 1997, Biophys Chem 68:9-16;Morea et al., 2000, Methods 20:267-279, and the conserved features ofantibodies are disclosed, for example, in Maynard et al., 2000, Annu RevBiomed Eng 2:339-376, both incorporated entirely by reference.

Antibodies are grouped into classes, also referred to as isotypes, asdetermined genetically by the constant region. Human constant lightchains are classified as kappa (Cκ) and lambda (Cλ) light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. TheIgG class is the most commonly used for therapeutic purposes. In humansthis class comprises subclasses IgG1, IgG2, IgG3, and IgG4. In mice thisclass comprises subclasses IgG1, IgG2a, IgG2b, IgG3. IgM has subclasses,including, but not limited to, IgM1 and IgM2. IgA has severalsubclasses, including but not limited to IgA1 and IgA2. Thus, “isotype”as used herein is meant any of the classes or subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. The known human immunoglobulin isotypes areIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. FIG. 2provides the sequences of the human light chain kappa and heavy chaingamma constant chains. FIG. 3 shows an alignment of the human IgGconstant heavy chains.

Also useful for the invention may be IgGs that are hybrid compositionsof the natural human IgG isotypes. Effector functions such as ADCC,ADCP, CDC, and serum half-life differ significantly between thedifferent classes of antibodies, including for example, human IgG1,IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgG, and IgM (Michaelsen et al.,1992, Molecular Immunology, 29(3): 319-326, entirely incorporated byreference). A number of studies have explored IgG1, IgG2, IgG3, and IgG4variants in order to investigate the determinants of the effectorfunction differences between them. See for example Canfield & Morrison,1991, J. Exp. Med. 173: 1483-1491; Chappel et al., 1991, Proc. Natl.Acad. Sci. USA 88(20): 9036-9040; Chappel et al., 1993, Journal ofBiological Chemistry 268:25124-25131; Tao et al., 1991, J. Exp. Med.173: 1025-1028; Tao et al., 1993, J. Exp. Med. 178: 661-667; Redpath etal., 1998, Human Immunology, 59, 720-727, all entirely incorporated byreference.

As described in U.S. Ser. No. 11/256,060, filed Oct. 21, 2005, entitled“IgG Immunoglobulin Variants with Optimized Effector Function”, hereinexpressly incorporated by reference, it is possible to engineer aminoacid modifications in an antibody that comprise constant regions fromother immunoglobulin classes, for example as those illustrated in thealignments in FIG. 3. Such engineered hybrid IgG compositions mayprovide improved effector function properties, including improved ADCC,phagocytosis, CDC, and serum half-life. For example, as illustrated byFIG. 3, an IgG1/IgG3 hybrid variant may be constructed by substitutingIgG1 positions in the CH2 and CH3 region with the amino acids from IgG3at positions where the two isotypes differ. Thus a hybrid variant IgGantibody may be constructed that comprises one or more substitutionsselected from the group consisting of: 274Q, 276K, 300F, 339T, 356E,358M, 384S, 392N, 397M, 422I, 435R, and 436F, wherein numbering isaccording to the EU index. Such variant may provide alternate and/orimproved effector function properties.

As another example, relatively poor effector function of IgG2 may beimproved by replacing key FcγR binding residues with the correspondingamino acids in an IgG with better effector function. For example, keyresidue differences between IgG2 and IgG1 with respect to FcγR bindingmay include P233, V234, A235, -236 (referring to a deletion in IgG2relative to IgG1), and G327. Thus one or more amino acid modificationsin the parent IgG2 wherein one or more of these residues is replacedwith the corresponding IgG1 amino acids, P233E, V234L, A235L, -236G(referring to an insertion of a glycine at position 236), and G327A, mayprovide enhanced effector function. The sequence of such an IgG,comprising a hybrid of residues from IgG1 and IgG2, refered to herein as“Hybrid” in the Examples and Figures, is provided in FIG. 2.

As is well known in the art, immunoglobulin polymorphisms exist in thehuman population. Gm polymorphism is determined by the IGHG1, IGHG2 andIGHG3 genes which have alleles encoding allotypic antigenic determinantsrefered to as G1m, G2m, and G3m allotypes for markers of the human IgG1,IgG2 and IgG3 molecules (no Gm allotypes have been found on the gamma 4chain). Markers may be classified into ‘allotypes’ and ‘isoallotypes’.These are distinguished on different serological bases dependent uponthe strong sequence homologies between isotypes. Allotypes are antigenicdeterminants specified by allelic forms of the Ig genes. Allotypesrepresent slight differences in the amino acid sequences of heavy orlight chains of different individuals. Even a single amino aciddifference can give rise to an allotypic determinant, although in manycases there are several amino acid substitutions that have occurred.Allotypes are sequence differences between alleles of a subclass wherebythe antisera recognize only the allele differences. An isoallotype is anallele in one isotype which produces an epitope which is shared with anon-polymorphic homologous region of one or more other isotypes andbecause of this the antisera will react with both the relevant allotypesand the relevant homologous isotypes (Clark, 1997, IgG effectormechanisms, Chem Immunol. 65:88-110; Gorman & Clark, 1990, Semin Immunol2(6):457-66, both incorporated entirely by reference).

Allelic forms of human immunoglobulins have been well-characterized (WHOReview of the notation for the allotypic and related markers of humanimmunoglobulins. J Immunogen 1976, 3: 357-362; WHO Review of thenotation of the allotypic and related markers of human immunoglobulins,1976, Eur. J. Immunol. 6, 599-601; E. van Loghem, 1986, Allotypicmarkers, Monogr Allergy 19: 40-51, all incorporated entirely byreference). Additionally, other polymorphisms have been characterized(Kim et al., 2001, J. Mol. Evol. 54:1-9, incorporated entirely byreference). At present, 18 Gm allotypes are known: G1m (1, 2, 3, 17) orG1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15,16, 21, 24, 26, 27, 28) or G3m (b1, c3, b3, b0, b3, b4, s, t, g1, c5, u,v, g5) (Lefranc, et al., The human IgG subclasses: molecular analysis ofstructure, function and regulation. Pergamon, Oxford, pp. 43-78 (1990);Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211, both incorporatedentirely by reference). Allotypes that are inherited in fixedcombinations are called Gm haplotypes. FIG. 4 shows common haplotypes ofthe gamma chain of human IgG1 (FIG. 4a ) and IgG2 (FIG. 4b ) showing thepositions and the relevant amino acids substitutions. Amino acidsequences of these allotypic versions of IgG1 and IgG2 are provided asSEQ IDs: 80-85. The antibodies of the present invention may besubstantially encoded by any allotype, isoallotype, or haplotype of anyimmunoglobulin gene.

Allelic forms of human immunoglobulins have been well-characterized (WHOReview of the notation for the allotypic and related markers of humanimmunoglobulins. J Immunogen 1976, 3: 357-362; WHO Review of thenotation of the allotypic and related markers of human immunoglobulins,1976, Eur. J. Immunol. 6, 599-601; E. van Loghem, 1986, Allotypicmarkers, Monogr Allergy 19: 40-51, all incorporated entirely byreference). Additionally, other polymorphisms have been characterized(Kim et al., 2001, J. Mol. Evol. 54:1-9, incorporated entirely byreference). At present, 18 Gm allotypes are known: G1m (1, 2, 3, 17) orG1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15,16, 21, 24, 26, 27, 28) or G3m (b1, c3, b3, b0, b3, b4, s, t, g1, c5, u,v, g5) (Lefranc, et al., The human IgG subclasses: molecular analysis ofstructure, function and regulation. Pergamon, Oxford, pp. 43-78 (1990);Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211, both incorporatedentirely by reference). Allotypes that are inherited in fixedcombinations are called Gm haplotypes. FIG. 4 shows common haplotypes ofthe gamma chain of human IgG1 (FIG. 4a ) and IgG2 (FIG. 4b ) showing thepositions and the relevant amino acid substitutions. The antibodies ofthe present invention may be substantially encoded by any allotype,isoallotype, or haplotype of any immunoglobulin gene.

Antibodies of the present invention may be substantially encoded bygenes from any organism, preferably mammals, including but not limitedto humans, rodents including but not limited to mice and rats,lagomorpha including but not limited to rabbits and hares, camelidaeincluding but not limited to camels, llamas, and dromedaries, andnon-human primates, including but not limited to Prosimians, Platyrrhini(New World monkeys), Cercopithecoidea (Old World monkeys), andHomimoidea including the Gibbons and Lesser and Great Apes. In a mostpreferred embodiment, the antibodies of the present invention aresubstantially human. The antibodies of the present invention may besubstantially encoded by immunoglobulin genes belonging to any of theantibody classes. In a most preferred embodiment, the antibodies of thepresent invention comprise sequences belonging to the IgG class ofantibodies, including human subclasses IgG1, IgG2, IgG3, and IgG4. In analternate embodiment, the antibodies of the present invention comprisesequences belonging to the IgA (including human subclasses IgA1 andIgA2), IgD, IgE, IgG, or IgM classes of antibodies. The antibodies ofthe present invention may comprise more than one protein chain. That is,the present invention may find use in an antibody that is a monomer oran oligomer, including a homo- or hetero-oligomer.

In the most preferred embodiment, the antibodies of the invention arebased on human IgG sequences, and thus human IgG sequences are used asthe “base” sequences against which other sequences are compared,including but not limited to sequences from other organisms, for examplerodent and primate sequences, as well as sequences from otherimmunoglobulin classes such as IgA, IgE, IgGD, IgGM, and the like. It iscontemplated that, although the antibodies of the present invention areengineered in the context of one parent antibody, the variants may beengineered in or “transferred” to the context of another, second parentantibody. This is done by determining the “equivalent” or“corresponding” residues and substitutions between the first and secondantibodies, typically based on sequence or structural homology betweenthe sequence of the two antibodies. In order to establish homology, theamino acid sequence of a first antibody outlined herein is directlycompared to the sequence of a second antibody. After aligning thesequences, using one or more of the homology alignment programs known inthe art (for example using conserved residues as between species),allowing for necessary insertions and deletions in order to maintainalignment (i.e., avoiding the elimination of conserved residues througharbitrary deletion and insertion), the residues equivalent to particularamino acids in the primary sequence of the first antibody are defined.Alignment of conserved residues preferably should conserve 100% of suchresidues. However, alignment of greater than 75% or as little as 50% ofconserved residues is also adequate to define equivalent residues.Equivalent residues may also be defined by determining structuralhomology between a first and second antibody that is at the level oftertiary structure for antibodies whose structures have been determined.In this case, equivalent residues are defined as those for which theatomic coordinates of two or more of the main chain atoms of aparticular amino acid residue of the parent or precursor (N on N, CA onCA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm afteralignment. Alignment is achieved after the best model has been orientedand positioned to give the maximum overlap of atomic coordinates ofnon-hydrogen protein atoms of the proteins. Regardless of how equivalentor corresponding residues are determined, and regardless of the identifyof the parent antibody in which the antibodies are made, what is meantto be conveyed is that the antibodies discovered by the presentinvention may be engineered into any second parent antibody that hassignificant sequence or structured homology with said antibody. Thus forexample, if a variant antibody is generated wherein the parent antibodyis human IgG1, by using the methods described above or other methods fordetermining equivalent residues, said variant antibody may be engineeredin a human IgG2 parent antibody, a human IgA parent antibody, a mouseIG2a or IgG2b parent antibody, and the like. Again, as described above,the context of the parent antibody does not affect the ability totransfer the antibodies of the present invention to other parentantibodies. For example, the variant antibodies that are engineered in ahuman IgG1 antibody that targets one antigen epitope may be transferredinto a human IgG2 antibody that targets a different antigen epitope, andso forth.

In the IgG class of immunoglobulin, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the domains of theconstant heavy chain, including, the constant heavy (CH) domains and thehinge. In the context of IgG antibodies, the IgG isotypes each havethree CH regions: “CH1” refers to positions 118-220, “CH2” refers topositions 237-340, and “CH3” refers to positions 341-447 according tothe EU index as in Kabat. By “hinge” or “hinge region” or “antibodyhinge region” or “immunoglobulin hinge region” herein is meant theflexible polypeptide comprising the amino acids between the first andsecond constant domains of an antibody. Structurally, the IgG CH1 domainends at EU position 220, and the IgG CH2 domain begins at residue EUposition 237. Thus for IgG the hinge is herein defined to includepositions 221 (D221 in IgG1) to 236 (G236 in IgG1), wherein thenumbering is according to the EU index as in Kabat. In some embodiments,for example in the context of an Fc region, the lower hinge is included,with the “lower hinge” generally referring to positions 226 or 230. Theconstant heavy chain, as defined herein, refers to the N-terminus of theCH1 domain to the C-terminus of the CH3 domain, thus comprisingpositions 118-447, wherein numbering is according to the EU index. Theconstant light chain comprises a single domain, and as defined hereinrefers to positions 108-214 of Cκ or Cλ, wherein numbering is accordingto the EU index.

Antibodies of the invention may include multispecific antibodies,notably bispecific antibodies, also sometimes referred to as“diabodies”. These are antibodies that bind to two (or more) differentantigens. Diabodies can be manufactured in a variety of ways known inthe art, e.g., prepared chemically or from hybrid hybridomas. In oneembodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scFv joined to a CH3 domain. In somecases, the scFV can be joined to the Fc region, and may include some orall of the hinge region. For a description of multispecific antibodiessee Holliger & Hudson, 2006, Nature Biotechnology 23(9):1126-1136 andreferences cited therein, all expressly incorporated by reference.

In one embodiment, the antibody of the invention is an antibodyfragment. Of particular interest are antibodies that comprise Fcregions, Fc fusions, and the constant region of the heavy chain(CH1-hinge-CH2-CH3). Antibodies of the present invention may comprise Fcfragments. An Fc fragment of the present invention may comprise from1-90% of the Fc region, with 10-90% being preferred, and 30-90% beingmost preferred. Thus for example, an Fc fragment of the presentinvention may comprise an IgG1 Cγ2 domain, an IgG1 Cγ2 domain and hingeregion, an IgG1 Cγ3 domain, and so forth. In one embodiment, an Fcfragment of the present invention additionally comprises a fusionpartner, effectively making it an Fc fragment fusion. Fc fragments mayor may not contain extra polypeptide sequence.

Chimeric, Humanized, and Fully Human Antibodies

Immunogenicity is the result of a complex series of responses to asubstance that is perceived as foreign, and may include production ofneutralizing and non-neutralizing antibodies, formation of immunecomplexes, complement activation, mast cell activation, inflammation,hypersensitivity responses, and anaphylaxis. Several factors cancontribute to protein immunogenicity, including but not limited toprotein sequence, route and frequency of administration, and patientpopulation. Immunogenicity may limit the efficacy and safety of aprotein therapeutic in multiple ways. Efficacy can be reduced directlyby the formation of neutralizing antibodies. Efficacy may also bereduced indirectly, as binding to either neutralizing ornon-neutralizing antibodies typically leads to rapid clearance fromserum. Severe side effects and even death may occur when an immunereaction is raised. Thus in a preferred embodiment, protein engineeringis used to reduce the immunogenicity of the antibodies of the presentinvention.

In some embodiments, the scaffold components can be a mixture fromdifferent species. Such antibody may be a chimeric antibody and/or ahumanized antibody. In general, both “chimeric antibodies” and“humanized antibodies” refer to antibodies that combine regions frommore than one species. “Chimeric antibodies” traditionally comprisevariable region(s) from a mouse (or rat, in some cases) and the constantregion(s) from a human (Morrison et al., 1984, Proc Natl Acad Sci USA81: 6851-6855, incorporated entirely by reference).

By “humanized” antibody as used herein is meant an antibody comprising ahuman framework region (FR) and one or more complementarity determiningregions (CDR's) from a non-human (usually mouse or rat) antibody. Thenon-human antibody providing the CDR's is called the “donor” and thehuman immunoglobulin providing the framework is called the “acceptor”.In certain embodiments, humanization relies principally on the graftingof donor CDRs onto acceptor (human) VL and VH frameworks (Winter U.S.Pat. No. 5,225,539, incorporated entirely by reference). This strategyis referred to as “CDR grafting”. “Backmutation” of selected acceptorframework residues to the corresponding donor residues is often requiredto regain affinity that is lost in the initial grafted construct (U.S.Pat. No. 5,693,762, incorporated entirely by reference). The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region, typically that of a humanimmunoglobulin, and thus will typically comprise a human Fc region. Avariety of techniques and methods for humanizing and reshaping non-humanantibodies are well known in the art (See Tsurushita & Vasquez, 2004,Humanization of Monoclonal Antibodies, Molecular Biology of B Cells,533-545, Elsevier Science (USA), and references cited therein, allincorporated entirely by reference). Humanization or other methods ofreducing the immunogenicity of nonhuman antibody variable regions mayinclude resurfacing methods, as described for example in Roguska et al.,1994, Proc. Natl. Acad. Sci. USA 91:969-973, incorporated entirely byreference. In one embodiment, selection based methods may be employed tohumanize and/or affinity mature antibody variable regions, that is, toincrease the affinity of the variable region for its target antigen.Other humanization methods may involve the grafting of only parts of theCDRs, including but not limited to methods described in U.S. Ser. No.09/810,502; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084, incorporated entirely byreference. Structure-based methods may be employed for humanization andaffinity maturation, for example as described in U.S. Ser. No.10/153,159 and related applications, all incorporated entirely byreference.

In certain variations, the immunogenicity of the antibody is reducedusing a method described in U.S. Ser. No. 11/004,590, entitled “Methodsof Generating Variant Proteins with Increased Host String Content andCompositions Thereof”, filed on Dec. 3, 2004, incorporated entirely byreference.

Modifications to reduce immunogenicity may include modifications thatreduce binding of processed peptides derived from the parent sequence toMHC proteins. For example, amino acid modifications would be engineeredsuch that there are no or a minimal number of immune epitope that arepredicted to bind, with high affinity, to any prevalent MHC alleles.Several methods of identifying MHC-binding epitopes in protein sequencesare known in the art and may be used to score epitopes in an antibody ofthe present invention. See for example U.S. Ser. No. 09/903,378, U.S.Ser. No. 10/754,296, U.S. Ser. No. 11/249,692, and references citedtherein, all expressly incorporated by reference.

In an alternate embodiment, the antibodies of the present invention maybe fully human, that is the sequences of the antibodies are completelyor substantially human. “Fully human antibody” or “complete humanantibody” refers to human antibody having the gene sequence of anantibody derived from a human chromosome with the modifications outlinedherein. A number of methods are known in the art for generating fullyhuman antibodies, including the use of transgenic mice (Bruggemann etal., 1997, Curr Opin Biotechnol 8:455-458,) or human antibody librariescoupled with selection methods (Griffiths et al., 1998, Curr OpinBiotechnol 9:102-108,) both incorporated entirely by reference.

Antibodies that Target CD19

The antibodies of the present invention may be virtually any antibodythat binds to CD19. The variable regions of any known or undiscoveredanti-CD19 antibodies may find use in the present invention. Antibodiesof the invention may display selectivity for CD19 versus alternativetargets, or selectivity for specific form of the target versusalternative forms. Examples include full-length versus splice variants,cell-surface vs. soluble forms, selectivity for various polymorphicvariants, or selectivity for specific conformational forms of a target.An antibody of the present invention may bind any epitope or region onCD19, and may be specific for fragments, mutant forms, splice forms, oraberrent forms of said antigens. A number of useful antibodies have beendiscovered that target CD19 that may find use in the present invention.Suitable antibodies or immunoadhesins include the CD19 antibodies orimmunoadhesins in MT-103 (a single-chain bispecific CD19/CD3 antibody;Hoffman, P. et al. 2005, Int. J. Cancer. 115: 98-104; Schlereth, B. etal. 2006, Cancer Immunol. Immunother. 55: 503-514), a CD19/CD16 disbody(Schlenzka, J. et al. 2004, Anti-cancer Drugs, 15: 915-919; Kipriyanov,S. M. et al. 2002, J. Immunol. 159: 137-144), BU12-saporin (Flavell, D.J. et al. 1995, Br. J. Cancer, 72: 1373-1379), and anti-CD19-idarubicin(Rowland, A. J. et al. 1993, Cancer Immunol. Immunother. 55: 503-514);Olson, US Pub. No. 2004/0136908A1, filed Mar. 4, 2004; U.S. Pat. No.5,686,072; Olson, WO 02/080987A1, filed Mar. 29, 2002; Tedder, WO06/133450A1, filed Jun. 8, 2006; Tedder, WO 06/121852A2, filed Arp. 5,2006; Tedder, WO 06/089133A2, filed Feb. 15, 2006; Tedderm US Pub. No.2006/280738A1, filed Jun. 8, 2006; U.S. Pat. No. 7,109,304; Hansen, USPub. No. 2005/070693A1, filed Aug. 2, 2004; Hansen, US Pub. No.2006/257398A1, filed Jun. 1, 2006; Hansen, WO 06/012493A2, filed Aug. 2,2004; Rao-Naik, WO 07/002223A2, filed Jun. 20, 2006; Page, US Pub.2002/182208A1, filed May 16, 2002; U.S. Pat. No. 5,686,072; Page,EP00481790B1, filed Oct. 17, 1991; Hariharan, US Pub. No. 2003/103971A1,filed Sep. 12, 2002; Goldenberg, US Pub. No. 2003/133930A1, filed Jan.24, 2003; Goldenberg, US Pub. No. 2004/219156A1, filed Dec. 30, 2002;Hariharan, US Pub. No. 2007/0009519A1, filed Jul. 21, 2006; Curd, WO00/067796A1, filed May 4, 2000; Kipriyanov, WO 03/088998A1, filed Arp.15, 2003; U.S. Pat. No. 7,112,324, U.S. Pat. No. 7,129,330, Olson, USPub. No. 2004/0136908A1, filed Mar. 4, 2004; Dorken, US Pub. No.2006/0193852A1, filed May 5, 2006; Amphlett, US Pub. No. 2007/0009541A1,filed Sep. 14, 2006; Kersey, WO 96/36360A1, filed May 15, 1996; Kufer,WO 04/106381A1, filed May 26, 2004; Little, US Pub. No. 2007/031436A1,filed Oct. 10, 2006; Kufer, US Pub. No. 2007/123479A1, filed May 26,2004; Baeuerle, WO 07/068354A1, filed Nov. 29, 2006; Le Gall, EP01314741B1, filed Nov. 14, 2001; Pesando, WO 91/13974A1, filed Mar. 12,1991; Allen et al, Clin. Cancer. Res 2005; 11(9) May 1, 2005; Barbin etal, J. Immunother, Vol. 29, No. 2, March/April 2006; Bruenke et al,Brit. J. Haem, 130, 218-2228 (2005); Callard, J. Immunol. Vol. 148,2983-1987, No. 10, May 15, 1992; Carter et al, Immunol. Res.2002/25/1-3:45-54; Carter & Barrington, Curr. Dir. Autoimmun. Basel,Karger, 2004, vol 7, pp 4-32; WWWK Cheng et al, Biochim. Biophys. Acta1768 (2007) 21-29; Cochlovius, Cancer Res. 60, 4336-4341, Aug. 15, 2000;LJN Cooper et al, Blood Cells, Molecules & Diseases, 33 (2004) 83-89;LIN Cooper et al, Blood, 15 Feb. 2005, Vol. 105, No. 4, pp 1622-1631;Culton et al, J. Clin. Immunol., Vol. 27, No. 1, January 2007; Daniel etal, Blood, Vol. 92, No. 12 (December 15), 1998, pp 4750-4757; Doody etal, Curr. Opin. Immun., 1996, pp 378-382; Dreier et al, Int. J. Cancer,100, 690-697 (2002); Dreier et al, J. Immunol., 2003, pp. 4397-4402;Featon & Carter, Annu. Rev. Immunol. 1995, 13:127-149; Fearon & Carroll,Annu. Rev. Immunol. 2000, 18:393-422; Fujimoto & Sato, J. Dermatol. Sci.(2007) in press; Le Gall et al, Prot. Engr. Des. & Select., vol. 17, no.4, pp. 357-366, 2004; Ghetie et al, Blood, 1 Jul. 2004, Vol. 104, No. 1,pp. 178-183; Ghetie et al, Blood, Vol. 83, No. 5 (March 1), 1994; pp1329-1336; Ghetie et al, Clin. Cancer Res., Vol. 5, 3920-2927, December1999; Ginaldi et al, J. Clin. Pathol, 1998; 51:364-369; Grossbard et al,Clin. Cancer Res., Vol. 5, 2392-2398, September 1999; Grossbard et al,Brit. J. Haematol., 1998, 102, 509-515; Grossbard et al, Blood, Vol. 80,No. 4 (August 15), 1992; pp 863-878; Grossbard & Fidias, Clin. Immunol.& Immunolpath., Vol. 76, No. 2, August, pp. 107-114, 1995; M. Green,Cancer Immunol. Immunother. (2004) 53: 625-632; Harata et al, Blood, 1Sep. 1004, Vol. 104, No. 5, pp 1442-1449; Hekman et al, Cancer Immunol.Immunother. (1991) 32: 364-372; Hoffmann et al, Int. J. Cancer, 115,98-104 (2005); Kipriyanov et al, J. Immunol. (2002), pp. 138-144;Kipriyanov et al, Int. J. Cancer. 77, 763-772 (1998); Kipriyanov et al,J. Immunol. Meth 196 (1996) 51-62; Lang et al, Blood, 15 May 2004, Vol.103, No. 10, pp 3982-3985; Lankester et al, J. Biol. Chem., Vol. 271,No. 37, September 13, pp. 22326-22330, 1996; Loeffler et al, Blood, 15Mar. 2000, Vol. 95, No. 6, pp 2098-2103; Masir, et al Histopathol.,2006, 48, pps. 239-246; Bargou et al, MT103 (MEDI-538) Poster; Mitchellet al, J. Nucl. Med. 2003, 44:1106-1112; Molhoj et al, Molec. Immunol,44 (2007) 1935-1943; Pietersz et al, Cancer Immunol. Immunother (1995)41: 53-60; Sapra et al, Clin. Cancer Res Vol. 10, 1100-1111, Feb. 1,2004; Schlereth et al, Cancer Immunol. Immunother. (2006) 53: 503-514;Schwemmlein et al, Leukemia (2007) 21, 1405-1412; Sieber et al, Brit. J.Haematology, 2003, 121, 458-461; Stone et al, Blood, Vol. 88, no. 4(August 15), 1996: 1188-1197; Sun et al, Molec. Immunolog, 41 (2004)929-938; Tedder & Isaacs, J. Immunolog, Vol. 143, 712-717, No. 2 Jul.15, 1989; Tedder et al, Curr. Dir. Autoimmun. Basel, Karger, 2005, vol8, pp 55-90; Tedder et al, Springer Semin. Immun. (2006) 28:351-364;Tiroch et al, J. Immunol., 2002, 168, 3275-3282; Uckun et al, Blood, Vol71, No 1 (January), 1988: pp 13-29; Uckun et al, J. Immunol, Vol 134, No3, March 1985, pp 2010-2016; Vallera et al, Clin. Cancer Res. 2005;11(10) May 16, 2005; Vlasveld et al, Cancer Immunol. Immunother (1995)40: 37-47; Vuist et al, Cancer Res, 49, 3783-3788, Jul. 15, 1989; Vuiset al, Cancer Res. 50, 5767-5772, Sep. 15, 1990; Yan et al, Int.Immunol. Vol 17, No. 7, pp 869-877 (2005); Yazawa, et la, PNAS 2005;102; 15178-15183, all hereby incorporated entirely by reference. Themolecules described in U.S. Pat. No. 5,686,072, WO 02/080987A1 and USPub. No. 2004/0136908A1 and identified as 4G7, the molecules describedin WO 1007/002223A2 and Tedder, are preferred.

The antibodies of the present invention may find use in a wide range ofproducts. In one embodiment the antibody of the invention is atherapeutic, a diagnostic, or a research reagent, preferably atherapeutic. Alternatively, the antibody of the present invention may beused for agricultural or industrial uses. An antibody of the presentinvention may find use in an antibody composition that is monoclonal orpolyclonal. The antibodies of the present invention may be agonists,antagonists, neutralizing, inhibitory, or stimulatory. In a preferredembodiment, the antibodies of the present invention are used to killtarget cells that bear the target antigen, for example cancer cells. Inan alternate embodiment, the antibodies of the present invention areused to block, antagonize, or agonize the target antigen. In analternately preferred embodiment, the antibodies of the presentinvention are used to block, antagonize, or agonize the target antigenand kill the target cells that bear the target antigen.

Anti-CD19 Antibodies as Therapeutics to Treat B-cell Disorders

Antibodies are a class of therapeutic proteins that may be used to treatB-cell disorders. A number of favorable properties of antibodies,including but not limited to specificity for target, ability to mediateimmune effector mechanisms, and long half-life in serum, make antibodiespowerful therapeutics. The present invention describes antibodiesagainst the B-cell antigen CD19.

B-cell antigen CD19 (CD19, also known as B-cell surface antigen B4,Leu-12) is a human pan-B-cell surface marker that is expressed fromearly stages of pre-B cell development through terminal differentiationinto plasma cells. CD19 promotes the proliferation and survival ofmature B cells. It associates in a complex with CD21 on the cellsurface. It also associates with CD81 and Leu-13 and potentiates B cellreceptor (BCR) signaling. Together with the BCR, CD19 modulatesintrinsic and antigen receptor-induced signaling thresholds critical forclonal expansion of B cells and humoral immunity. In collaboration withCD21 it links the adaptive and the inmate immune system. Uponactivation, the cytoplasmic tail of CD19 becomes phosphorylated whichleads to binding by Src-family kinases and recruitment of PI-3 kinase.It is an attractive immunotherapy target for cancers of lymphoid originsince it is also expressed on the vast majority of NHL cells as well assome leukemias.

A number of antibodies or antibody conjugates that target CD19 have beenevaluated in pre-clinical studies or in clinical trials for thetreatment of cancers. These anti-CD19 antibodies or antibody conjugatesinclude but are not limited to MT-103 (a single-chain bispecificCD19/CD3 antibody; Hoffman et al, 2005 Int J Cancer 115:98-104;Schlereth et al, 2006 Cancer Immunol Immunother 55:503-514), a CD19/CD16diabody (Schlenzka et al, 2004 Anti-cancer Drugs 15:915-919; Kipriyanovet al, 2002 J Immunol 169:137-144), BU12-saporin (Flavell et al, 1995 BrJ Cancer 72:1373-1379), and anti-CD19-idarubicin (Rowland et al, 1993Cancer Immunol Immunother 55:503-514); all expressly incorporated byreference.

Fc Optimization of Anti-CD19 Antibodies

There are a number of characterized mechanisms by which antibodiesmediate cellular effects, including anti-proliferation via blockage ofneeded growth pathways, intracellular signaling leading to apoptosis,enhanced down regulation and/or turnover of receptors,complement-dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediatedphagocytosis (ADCP) and promotion of an adaptive immune response (Cragget al., 1999, Curr Opin Immunol 11:541-547; Glennie et al., 2000,Immunol Today 21:403-410, both incorporated entirely by reference).Antibody efficacy may be due to a combination of these mechanisms, andtheir relative importance in clinical therapy for oncology appears to becancer dependent.

The importance of FcγR-mediated effector functions for the activity ofsome antibodies has been demonstrated in mice (Clynes et al., 1998, ProcNatl Acad Sci USA 95:652-656; Clynes et al., 2000, Nat Med 6:443-446,both incorporated entirely by reference), and from observed correlationsbetween clinical efficacy in humans and their allotype of high (V158) orlow (F158) affinity polymorphic forms of FcγRIIIa (Cartron et al., 2002,Blood 99:754-758; Weng & Levy, 2003, Journal of Clinical Oncology,21:3940-3947, both incorporated entirely by reference). Together thesedata suggest that an antibody that is optimized for binding to certainFcγRs may better mediate effector functions, and thereby destroy targetcells more effectively in patients. Thus a promising means for enhancingthe anti-tumor potency of antibodies is via enhancement of their abilityto mediate cytotoxic effector functions such as ADCC, ADCP, and CDC.Additionally, antibodies can mediate anti-tumor mechanism via growthinhibitory or apoptotic signaling that may occur when an antibody bindsto its target on tumor cells. Such signaling may be potentiated whenantibodies are presented to tumor cells bound to immune cells via FcγR.Therefore increased affinity of antibodies to FcγRs may result inenhanced anti-proliferative effects.

Antibody engineering for optimized effector function has been achievedusing amino acid modifications (see for example U.S. Ser. No. 10/672,280and U.S. Ser. No. 11/124,620 and references cited therein, allincorporated entirely by reference), and engineered glycoforms (see forexample Umaña et al., 1999, Nat Biotechnol 17:176-180; Shinkawa et al.,2003, J Biol Chem 278:3466-3473, Yamane-Ohnuki et al., 2004,Biotechnology and Bioengineering 87(5):614-621, all incorporatedentirely by reference).

Modifications for Optimizing Effector Function

The present invention is directed to antibodies comprisingmodifications, wherein said modifications alter affinity to one or moreFc receptors, and/or alter the ability of the antibody to mediate one ormore effector functions. Modifications of the invention include aminoacid modifications and glycoform modifications.

Amino Acid Modifications

As described in U.S. Ser. No. 11/124,620, filed May 5, 2005, entitled“Optimized Fc Variants”, and incorporated entirely by reference, aminoacid modifications at heavy chain constant region positions 221, 222,223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 243, 244, 245, 246, 247, 249, 255, 257, 260, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278,280, 281, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 313, 317, 318,320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335, 336, and 337, allow modification of Fc65R binding properties,effector function, and potentially clinical properties of antibodies.

In particular, variants that alter binding to one or more Fc receptorsmay comprise an amino acid modification in the heavy chain constantregion, as described herein, selected from the group consisting of 221K,221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E, 227G,227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231E, 231G,231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H,233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233Y,234A, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234P, 234Q,234R, 234S, 234T, 234V, 234W, 234Y, 235A, 235D, 235E, 235F, 235G, 235H,235I, 235K, 235M, 235N, 235P, 235Q, 235R, 235S, 235T, 235V, 235W, 235Y,236A, 236D, 236E, 236F, 236H, 236I, 236K, 236L, 236M, 236N, 236P, 236Q,236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H, 237I, 237K,237L, 237M, 237N, 237P, 237Q, 237R, 237S, 237T, 237V, 237W, 237Y, 238D,238E, 238F, 238G, 238H, 238I, 238K, 238L, 238M, 238N, 238Q, 238R, 238S,238T, 238V, 238W, 238Y, 239D, 239E, 239F, 239G, 239H, 239I, 239K, 239L,239M, 239N, 239P, 239Q, 239R, 239T, 239V, 239W, 239Y, 240A, 240I, 240M,240T, 241D, 241E, 241L, 241R, 241S, 241W, 241Y, 243E, 243H, 243L, 243Q,243R, 243W, 243Y, 244H, 245A, 246D, 246E, 246H, 246Y, 247G, 247V, 249H,249Q, 249Y, 255E, 255Y, 258H, 258S, 258Y, 260D, 260E, 260H, 260Y, 262A,262E, 262F, 262I, 262T, 263A, 263I, 263M, 263T, 264A, 264D, 264E, 264F,264G, 264H, 264I, 264K, 264L, 264M, 264N, 264P, 264Q, 264R, 264S, 264T,264W, 264Y, 265F, 265G, 265H, 265I, 265K, 265L, 265M, 265N, 265P, 265Q,265R, 265S, 265T, 265V, 265W, 265Y, 266A, 266I, 266M, 266T, 267D, 267E,267F, 267H, 267I, 267K, 267L, 267M, 267N, 267P, 267Q, 267R, 267T, 267V,267W, 267Y, 268D, 268E, 268F, 268G, 268I, 268K, 268L, 268M, 268P, 268Q,268R, 268T, 268V, 268W, 269F, 269G, 269H, 269I, 269K, 269L, 269M, 269N,269P, 269R, 269S, 269T, 269V, 269W, 269Y, 270F, 270G, 270H, 270I, 270L,270M, 270P, 270Q, 270R, 270S, 270T, 270W, 270Y, 271A, 271D, 271E, 271F,271G, 271H, 271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S, 271T, 271V,271W, 271Y, 272D, 272F, 272G, 272H, 272I, 272K, 272L, 272M, 272P, 272R,272S, 272T, 272V, 272W, 272Y, 273I, 274D, 274E, 274F, 274G, 274H, 274I,274L, 274M, 274N, 274P, 274R, 274T, 274V, 274W, 274Y, 275L, 275W, 276D,276E, 276F, 276G, 276H, 276I, 276L, 276M, 276P, 276R, 276S, 276T, 276V,276W, 276Y, 278D, 278E, 278G, 278H, 278I, 278K, 278L, 278M, 278N, 278P,278Q, 278R, 278S, 278T, 278V, 278W, 280G, 280K, 280L, 280P, 280W, 281D,281E, 281K, 281N, 281P, 281Q, 281Y, 282E, 282G, 282K, 282P, 282Y, 283G,283H, 283K, 283L, 283P, 283R, 283Y, 284D, 284E, 284L, 284N, 284Q, 284T,284Y, 285D, 285E, 285K, 285Q, 285W, 285Y, 286E, 286G, 286P, 286Y, 288D,288E, 288Y, 290D, 290H, 290L, 290N, 290W, 291D, 291E, 291G, 291H, 291I,291Q, 291T, 292D, 292E, 292T, 292Y, 293F, 293G, 293H, 293I, 293L, 293M,293N, 293P, 293R, 293S, 293T, 293V, 293W, 293Y, 294F, 294G, 294H, 294I,294K, 294L, 294M, 294P, 294R, 294S, 294T, 294V, 294W, 294Y, 295D, 295E,295F, 295G, 295H, 295I, 295M, 295N, 295P, 295R, 295S, 295T, 295V, 295W,295Y, 296A, 296D, 296E, 296G, 296H, 296I, 296K, 296L, 296M, 296N, 296Q,296R, 296S, 296T, 296V, 297D, 297E, 297F, 297G, 297H, 297I, 297K, 297L,297M, 297P, 297Q, 297R, 297S, 297T, 297V, 297W, 297Y, 298A, 298D, 298E,298F, 298H, 298I, 298K, 298M, 298N, 298Q, 298R, 298T, 298W, 298Y, 299A,299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M, 299N, 299P, 299Q,299R, 299S, 299V, 299W, 299Y, 300A, 300D, 300E, 300G, 300H, 300K, 300M,300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W, 301D, 301E, 301H, 301Y,302I, 303D, 303E, 303Y, 304D, 304H, 304L, 304N, 304T, 305E, 305T, 305Y,313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y, 320D, 320F, 320G, 320H,320I, 320L, 320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D, 322F, 322G,322H, 322I, 322P, 322S, 322T, 322V, 322W, 322Y, 323I, 324D, 324F, 324G,324H, 324I, 324L, 324M, 324P, 324R, 324T, 324V, 324W, 324Y, 325A, 325D,325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q, 325R, 325S,325T, 325V, 325W, 325Y, 326F, 326I, 326L, 326P, 326T, 327D, 327E, 327F,327H, 327I, 327K, 327L, 327M, 327N, 327P, 327R, 327S, 327T, 327V, 327W,327Y, 328A, 328D, 328E, 328F, 328G, 328H, 328I, 328K, 328M, 328N, 328P,328Q, 328R, 328S, 328T, 328V, 328W, 328Y, 329D, 329E, 329F, 329G, 329H,329I, 329K, 329L, 329M, 329N, 329Q, 329R, 329S, 329T, 329V, 329W, 329Y,330E, 330F, 330G, 330H, 330I, 330L, 330M, 330N, 330P, 330R, 330S, 330T,330V, 330W, 330Y, 331D, 331F, 331H, 331I, 331L, 331M, 331Q, 331R, 331T,331V, 331W, 331Y, 332A, 332D, 332E, 332F, 332H, 332K, 332L, 332M, 332N,332P, 332Q, 332R, 332S, 332T, 332V, 332W, 332Y, 333A, 333F, 333H, 333I,333L, 333M, 333P, 333T, 333Y, 334A, 334F, 334I, 334L, 334P, 334T, 335D,335F, 335G, 335H, 335I, 335L, 335M, 335N, 335P, 335R, 335S, 335V, 335W,335Y, 336E, 336K, 336Y, 337E, 337H, and 337N, wherein numbering isaccording to the EU index.

As described in U.S. Ser. No. 11/090,981, filed Mar. 24, 2005, entitled“Immunoglobulin variants outside the Fc region”, and incorporatedentirely by reference, amino acid modifications at heavy chain constantregion positions 118, 119, 120, 121, 122, 124, 126, 129, 131, 132, 133,135, 136, 137, 138, 139, 147, 148, 150, 151, 152, 153, 155, 157, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 183, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 201, 203, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, and 236, allow modificationof FcγR binding properties, effector function, and potentially clinicalproperties of antibodies.

As described in U.S. Ser. No. 11/090,981, filed Mar. 24, 2005, entitled“Immunoglobulin variants outside the Fc region”, and incorporatedentirely by reference, amino acid modifications at light chain constantregion positions 108, 109, 110, 111, 112, 114, 116, 121, 122, 123, 124,125, 126, 127, 128, 128, 131, 137, 138, 140, 141, 142, 143, 145, 147,149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 176, 180, 181,182, 183, 184, 185, 187, 188, 189, 190, 191, 193, 195, 197, 199, 200,202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, allowmodification of FcγR binding properties, effector function, andpotentially clinical properties of antibodies.

In particular, variants that alter binding to one or more human Fcreceptors may comprise an amino acid modification in the heavy chainconstant region, as described herein, selected from the group consistingof 118K, 118E, 118Y, 119R, 119E, 119Y, 120R, 120E, 120I, 121E, 121Y,121H, 122E, 122R, 124K, 124E, 124Y, 126K, 126D, 129L, 129D, 131G, 131T,132D, 132R, 132L, 133R, 133E, 133L, 135I, 135E, 135K, 136E, 136K, 136I,137E, 138S, 138R, 138D, 139I, 139E, 139K, 147A, 147E, 148Y, 148K, 150L,150K, 150E, 151A, 151D, 152L, 152K, 153L, 153D, 155E, 155K, 155I, 157E,157K, 157Y, 159K, 159D, 159L, 160K, 160E, 160Y, 161D, 162D, 162K, 162Y,163R, 164R, 164E, 164Y, 165D, 165R, 165Y, 166D, 167A, 168L, 169E, 171G,171H, 172K, 172L, 172E, 173T, 173D, 174E, 174K, 174Y, 175D, 175L, 176D,176R, 176L, 177R, 177E, 177Y, 178D, 179K, 179Y, 179E, 180K, 180L, 180E,183T, 187I, 187K, 187E, 188I, 189D, 189G, 190I, 190K, 190E, 191D, 191R,191Y, 192N, 192R, 192L, 193F, 193E, 194R, 194D, 195R, 195D, 195Y, 196K,196D, 196L, 197R, 197E, 197Y, 198L, 199T, 199D, 199K, 201E, 201K, 201L,203D, 203L, 203K, 205D, 205L, 206A, 206E, 207K, 207D, 208R, 208E, 208Y,209E, 209K, 209Y, 210L, 210E, 210Y, 211R, 211E, 211Y, 212Q, 212K, 212H,212L, 212Y, 213N, 213E, 213H, 213I, 213Y, 214N, 214E, 214H, 214L, 214Y,216N, 216K, 216H, 216L, 216Y, 217D, 217H, 217A, 217V, 217G, 218D, 218E,218Q, 218T, 218H, 218L, 218Y, 219D, 219E, 219Q, 219K, 219T, 219H, 219L,219I, 219Y, 204A, 210A, 213A, 214A, 218A, 221K, 221Y, 221E, 221N, 221Q,221R, 221S, 221T, 221H, 221A, 221V, 221L, 221I, 221F, 221M, 221W, 221P,221G, 222E, 222Y, 222D, 222N, 222Q, 222R, 222S, 222T, 222H, 222V, 222L,222I, 222F, 222M, 222W, 222P, 222G, 222A, 223D, 223N, 223Q, 223R, 223S,223H, 223A, 223V, 223L, 223I, 223F, 223M, 223Y, 223W, 223P, 223G, 223E,223K, 224D, 224N, 224Q, 224K, 224R, 224S, 224T, 224V, 224L, 224I, 224F,224M, 224W, 224P, 224G, 224E, 224Y, 224A, 225D, 225N, 225Q, 225R, 225S,225H, 225A, 225V, 225L, 225I, 225F, 225M, 225Y, 225P, 225G, 225E, 225K,225W, 226S, 227E, 227K, 227Y, 227G, 227D, 227N, 227Q, 227R, 227S, 227T,227H, 227A, 227V, 227L, 227I, 227F, 227M, 227W, 228K, 228Y, 228G, 228D,228N, 228Q, 228R, 228T, 228H, 228A, 228V, 228L, 228I, 228F, 228M, 228W,229S, 230A, 230E, 230Y, 230G, 230D, 230N, 230Q, 230K, 230R, 230S, 230T,230H, 230V, 230L, 230I, 230F, 230M, 230W, 231K, 231P, 231D, 231N, 231Q,231R, 231S, 231T, 231H, 231V, 231L, 231I, 231F, 231M, 231W, 232E, 232K,232Y, 232G, 232D, 232N, 232Q, 232R, 232S, 232T, 232H, 232A, 232V, 232L,232I, 232F, 232M, 232W, 233D, 233N, 233Q, 233R, 233S, 233T, 233H, 233A,233V, 233L, 233I, 233F, 233M, 233Y, 233W, 233G, 234D, 234E, 234N, 234Q,234T, 234H, 234Y, 234I, 234V, 234F, 234K, 234R, 234S, 234A, 234M, 234G,235D, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 235E, 235K,235R, 235A, 235M, 235W, 235P, 235G, 236D, 236E, 236N, 236Q, 236K, 236R,236S, 236T, 236H, 236A, 236V, 236L, 236I, 236F, 236M, 236Y, 236W, and236P, wherein numbering is according to the EU index.

In particular, variants that alter binding to one or more human Fcreceptors may comprise an amino acid modification in the light chainconstant region, as described herein, selected from the group consistingof 108D, 108I, 108Q, 109D, 109P, 109R, 110E, 110I, 110K, 111E, 111K,111L, 112E, 112R, 112Y, 114D, 114I, 114K, 116T, 121D, 122R, 122S, 122Y,123L, 123R, 124E, 125E, 125K, 126D, 126L, 126Q, 127A, 127D, 127K, 128N,129E, 129I, 129K, 131T, 137K, 137S, 138D, 138K, 138L, 140E, 140H, 140K,141E, 141K, 142D, 142G, 142L, 143A, 143L, 143R, 145D, 145T, 145Y, 147A,147E, 147K, 149D, 149Y, 150A, 151L, 151K, 152L, 152R, 152S, 153D, 153H,153S, 154E, 154R, 154V, 155E, 155I, 155K, 156A, 156D, 156R, 157N, 158D,158L, 158R, 159E, 159K, 159L, 160K, 160V, 161K, 161L, 162T, 163E, 163K,163T, 164Q, 165K, 165P, 165Y, 166E, 166M, 166S, 167K, 167L, 168K, 168Q,168Y, 169D, 160H, 169S, 170I, 170N, 170R, 171A, 171N, 171V, 172E, 172I,172K, 173K, 173L, 173Q, 174A, 176T, 180E, 180K, 180S, 181K, 182E, 182R,182T, 183D, 183L, 183P, 184E, 184K, 184Y, 185I, 185Q, 185R, 187K, 187Y,188E, 188S, 188Y, 189D, 189K, 189Y, 190E, 190L, 190R, 191E, 191R, 191S,193E, 193K, 193S, 195I, 195K, 195Q, 197E, 197K, 197L, 199E, 199K, 199Y,200S, 202D, 202R, 202Y, 203D, 203L, 203R, 204T, 205E, 205K, 206E, 206I,206K, 207A, 207E, 207L, 208E, 208K, 208T, 210A, 210E, 210K, 211A, 211E,211P, 212E, 212K, 212T, 213L, 213R, wherein numbering is according tothe EU index.

Additional substitutions that may also be used in the present inventioninclude other substitutions that modulate Fc receptor affinity,FcγR-mediated effector function, and/or complement mediated effectorfunction include but are not limited to 298A, 298T, 326A, 326D, 326E,326W, 326Y, 333A, 333S, 334L, and 334A (U.S. Pat. No. 6,737,056; Shieldset al, Journal of Biological Chemistry 2001, 276(9):6591-6604; U.S. Pat.No. 6,528,624; Idusogie et al., 2001, J. Immunology 166:2571-2572),247L, 255L, 270E, 392T, 396L, and 421K (U.S. Ser. No. 10/754,922; U.S.Ser. No. 10/902,588), and 280H, 280Q, and 280Y (U.S. Ser. No.10/370,749), all incorporated entirely by reference.

In other embodiments, antibodies of the present invention may becombined with constant heavy chain variants that alter FcRn binding.These include modifications that modify FcRn affinity in a pH-specificmanner. In particular, variants that increase Fc binding to FcRn includebut are not limited to: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton etal., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journalof Immunology 176:346-356, U.S. Ser. No. 11/102,621, PCT/US2003/033037,PCT/US2004/011213, U.S. Ser. No. 10/822,300, U.S. Ser. No. 10/687,118,PCT/US2004/034440, U.S. Ser. No. 10/966,673, all incorporated entirelyby reference), 256A, 272A, 286A, 305A, 307A, 311A, 312A, 376A, 378Q,380A, 382A, 434A (Shields et al, Journal of Biological Chemistry, 2001,276(9):6591-6004, U.S. Ser. No. 10/982,470, U.S. Pat. No. 5,737,056,U.S. Ser. No. 11/429,793, U.S. Ser. No. 11/429,786, PCT/US2005/029511,U.S. Ser. No. 11/208,422, all incorporated entirely by reference), 252F,252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S,433R, 433S, 433I, 433P, 433Q, 434H, 434F, 434Y, 252Y/254T/256E,433K/434F/436H, 308T/309P/311S (Dall Acqua et al. Journal of Immunology2002, 169:5171-5180, U.S. Pat. No. 7,083,784, PCT/US97/03321, U.S. Pat.No. 6,821,505, PCT/US01/48432, U.S. Ser. No. 11/397,328, allincorporated entirely by reference), 257C, 257M, 257L, 257N, 257Y, 279E,279Q, 279Y, insertion of Ser after 281, 283F, 284E, 306Y, 307V, 308P,308Y, 311V, 385H, 385N, (PCT/US2005/041220, U.S. Ser. No. 11/274,065,U.S. Ser. No. 11/436,266, all incorporated entirely by reference) 204D,284E, 285E, 286D, and 290E (PCT/US2004/037929 incorporated entirely byreference).

In some embodiments of the invention, antibodies may comprise isotypicmodifications, that is modifications in a parent IgG to the amino acidtype in an alternate IgG. For example as illustrated in FIG. 3, anIgG1/IgG3 hybrid variant may be constructed by substituting IgG1positions in the CH2 and/or CH3 region with the amino acids from IgG3 atpositions where the two isotypes differ. Thus a hybrid variant IgGantibody may be constructed that comprises one or more substitutionsselected from the group consisting of: 274Q, 276K, 300F, 339T, 356E,358M, 384S, 392N, 397M, 422I, 435R, and 436F. In other embodiments ofthe invention, an IgG1/IgG2 hybrid variant may be constructed bysubstituting IgG2 positions in the CH2 and/or CH3 region with aminoacids from IgG1 at positions where the two isotypes differ. Thus ahybrid variant IgG antibody may be constructed that comprises one ormore modifications selected from the group consisting of 233E, 234L,235L, -236G (referring to an insertion of a glycine at position 236),and 327A.

Glycoform Modifications

Many polypeptides, including antibodies, are subjected to a variety ofpost-translational modifications involving carbohydrate moieties, suchas glycosylation with oligosaccharides. There are several factors thatcan influence glycosylation. The species, tissue and cell type have allbeen shown to be important in the way that glycosylation occurs. Inaddition, the extracellular environment, through altered cultureconditions such as serum concentration, may have a direct effect onglycosylation. (Lifely et al., 1995, Glycobiology 5(8): 813-822),incorporated entirely by reference.

All antibodies contain carbohydrate at conserved positions in theconstant regions of the heavy chain. Each antibody isotype has adistinct variety of N-linked carbohydrate structures. Aside from thecarbohydrate attached to the heavy chain, up to 30% of human IgGs have aglycosylated Fab region. IgG has a single N-linked biantennarycarbohydrate at Asn297 of the CH2 domain. For IgG from either serum orproduced ex vivo in hybridomas or engineered cells, the IgG areheterogeneous with respect to the Asn297 linked carbohydrate (Jefferiset al., 1998, Immunol. Rev. 163:59-76; Wright et al., 1997, TrendsBiotech 15:26-32, both incorporated entirely by reference). For humanIgG, the core oligosaccharide normally consists of GlcNAc₂Man, GlcNAc,with differing numbers of outer residues.

The carbohydrate moieties of the present invention will be describedwith reference to commonly used nomenclature for the description ofoligosaccharides. A review of carbohydrate chemistry which uses thisnomenclature is found in Hubbard et al. 1981, Ann. Rev. Biochem.50:555-583, incorporated entirely by reference. This nomenclatureincludes, for instance, Man, which represents mannose; GlcNAc, whichrepresents 2-N-acetylglucosamine, Gal which represents galactose; Fucfor fucose; and Glc, which represents glucose. Sialic acids aredescribed by the shorthand notation NeuNAc, for 5-N-acetylneuraminicacid, and NeuNGc for 5-glycolylneuraminic.

The term “glycosylation” means the attachment of oligosaccharides(carbohydrates containing two or more simple sugars linked together e.g.from two to about twelve simple sugars linked together) to aglycoprotein. The oligosaccharide side chains are typically linked tothe backbone of the glycoprotein through either N- or O-linkages. Theoligosaccharides of the present invention occur generally are attachedto a CH2 domain of an Fc region as N-linked oligosaccharides. “N-linkedglycosylation” refers to the attachment of the carbohydrate moiety to anasparagine residue in a glycoprotein chain. The skilled artisan willrecognize that, for example, each of murine IgG1, IgG2a, IgG2b and IgG3as well as human IgG1, IgG2, IgG3, IgG4, IgA and IgD CH2 domains have asingle site for N-linked glycosylation at amino acid residue 297 (Kabatet al. Sequences of Proteins of Immunological Interest, 1991,incorporated entirely by reference).

For the purposes herein, a “mature core carbohydrate structure” refersto a processed core carbohydrate structure attached to an Fc regionwhich generally consists of the following carbohydrate structureGlcNAc(Fucose)-GlcNAc-Man-(Man-GlcNAc)₂ typical of biantennaryoligosaccharides. The mature core carbohydrate structure is attached tothe Fc region of the glycoprotein, generally via N-linkage to Asn297 ofa CH2 domain of the Fc region. A “bisecting GlcNAc” is a GlcNAc residueattached to the β1,4 mannose of the mature core carbohydrate structure.The bisecting GlcNAc can be enzymatically attached to the mature corecarbohydrate structure by a β(1,4)-N-acetylglucosaminyltransferase IIIenzyme (GnTIII). CHO cells do not normally express GnTIII (Stanley etal., 1984, J. Biol. Chem. 261:13370-13378), but may be engineered to doso (Umana et al., 1999, Nature Biotech. 17:176-180).

The present invention contemplates antibodies that comprise modifiedglycoforms or engineered glycoforms. By “modified glycoform” or“engineered glycoform” as used herein is meant a carbohydratecomposition that is covalently attached to a protein, for example anantibody, wherein said carbohydrate composition differs chemically fromthat of a parent protein. Engineered glycoforms may be useful for avariety of purposes, including but not limited to enhancing or reducingFcγR-mediated effector function. In a preferred embodiment, theantibodies of the present invention are modified to control the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region.

Historically, antibodies produced in Chinese Hamster Ovary Cells (CHO),one of the most commonly used industrial hosts, contain about 2 to 6% inthe population that are nonfucosylated. YB2/0 (rat myeloma) and Lec13cell line (a lectin mutant of CHO line which has a deficient GDP-mannose4,6 dehydratase leading to the deficiency of GDP-fucose or GDP-sugarintermediates that are the substrate of α1,6-fucosyltransferase (Ripkaet al., 1986), however, can produce antibodies with 78% to 98%nonfucosylated species. Unfortunately, the yield of antibody from thesecells is extremely poor and therefore these cell lines are not useful tomake therapeutic antibody products commercially. The FUT8 gene encodesthe α1,6-fucosyltransferase enzyme that catalyzes the tranfer of afucosyl residue from GDP-fucose to position 6 of Asn-linked (N-linked)GlcNac of an N-glycan (Yanagidani et al., 1997, J Biochem 121:626-632).It is known that the α1,6 fucosyltransferase is the only enzymeresponsible for adding fucose to the N-linked biantennary carbohydrateat Asn297 in the CH2 domain of the IgG antibody.

A variety of methods are well known in the art for generating modifiedglycoforms (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies etal., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473);(U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/133,929; PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCTWO 02/30954A1); Yamane-Ohnuki et al., 2004, Biotechnology andBioengineering 87(5):614-621; (Potelligent™ technology [Biowa, Inc.,Princeton, N.J.]; GlycoMAb™ glycosylation engineering technology[GLYCART biotechnology AG, Zürich, Switzerland]; all of which areexpressly incorporated by reference). These techniques control the levelof fucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines; engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells), by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed.

Other methods for modifying glycoforms of the antibodies of theinvention include using glycoengineered strains of yeast (Li et al.,2006, Nature Biotechnology 24(2):210-215), moss (Nechansky et al., 2007,Mol Immunol 44(7):1826-8), and plants (Cox et al., 2006, Nat Biotechnol24(12):1591-7). Methods for modifying glycoforms include but are notlimited to using a glycoengineered strain of yeast Pichia pastoris (Liet al., 2006, Nature Biotechnology 24(2):210-215), a glycoengineeredstrain of the moss Physcomitrella patens wherein the enzymesβ1,2-xylosyltransferase and/or α1,3-fucosyltransferase are knocked outin (Nechansky et al., 2007, Mol Immunjol 44(7):1826-8), and the use ofRNA interference to inhibit endogenous alpha-1,3-fucosyltransferaseand/or beta-1,2-xylosyltransferase in the aquatic plant Lemna minor (Coxet al., 2006, Nat Biotechnol 24(12):1591-7).

Modified or engineered glycoform typically refers to the differentcarbohydrate or oligosaccharide; thus for example an antibody maycomprise an engineered glycoform. Alternatively, engineered glycoformmay refer to the antibody that comprises the different carbohydrate oroligosaccharide. For the purposes of modified glycoforms describedherein, a “parent antibody” is a glycosylated antibody having the sameamino acid sequence and mature core carbohydrate structure as anengineered glycoform of the present invention, except that fucose isattached to the mature core carbohydrate structure of the parentantibody. For instance, in a composition comprising the parentglycoprotein about 50-100% or about 70-100% of the parent glycoproteincomprises a mature core carbohydrate structure having fucose attachedthereto.

The present invention provides a composition comprising a glycosylatedantibody having an Fc region, wherein about 51-100% of the glycosylatedantibody in the composition comprises a mature core carbohydratestructure which lacks fucose, attached to the Fc region of the antibody.More preferably, about 80-100% of the antibody in the compositioncomprises a mature core carbohydrate structure which lacks fucose andmost preferably about 90-99% of the antibody in the composition lacksfucose attached to the mature core carbohydrate structure. In a mostpreferred embodiment, the antibody in the composition both comprises amature core carbohydrate structure that lacks fucose and additionallycomprises at least one amino acid modification in the Fc region. In themost preferred embodiment, the combination of engineered glycoform andamino acid modification provides optimal Fc receptor binding propertiesto the antibody.

Optimized Properties of Antibodies

The present invention provides variant antibodies that are optimized fora number of therapeutically relevant properties. A variant antibodycomprises one or more amino acid modifications relative to a parentantibody, wherein said amino acid modification(s) provide one or moreoptimized properties. Thus the antibodies of the present invention arevariants antibodies. An antibody of the present invention differs inamino acid sequence from its parent antibody by virtue of at least oneamino acid modification. Thus variant antibodies of the presentinvention have at least one amino acid modification compared to theparent. Alternatively, the variant antibodies of the present inventionmay have more than one amino acid modification as compared to theparent, for example from about one to fifty amino acid modifications,preferably from about one to ten amino acid modifications, and mostpreferably about one to about five amino acid modifications compared tothe parent. Thus the sequences of the variant antibodies and those ofthe parent antibodies are substantially homologous. For example, thevariant antibody sequences herein will possess about 80% homology withthe parent antibody sequence, preferably at least about 90% homology,and most preferably at least about 95% homology.

In a most preferred embodiment, the antibodies of the present inventioncomprise amino acid modifications that provide optimized effectorfunction properties relative to the parent. Most preferred substitutionsand optimized effector function properties are described in U.S. Ser.No. 10/672,280, PCT US03/30249, and U.S. Ser. No. 10/822,31, and U.S.Ser. No. 60/627,774, filed Nov. 12, 2004 and entitled “Optimized FcVariants”. Properties that may be optimized include but are not limitedto enhanced or reduced affinity for an FcγR. In a preferred embodiment,the antibodies of the present invention are optimized to possessenhanced affinity for a human activating FcγR, preferably FcγRI,FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb, most preferably FcγRIIIa. Inan alternately preferred embodiment, the antibodies are optimized topossess reduced affinity for the human inhibitory receptor FcγRIIb.These preferred embodiments are anticipated to provide antibodies withenhanced therapeutic properties in humans, for example enhanced effectorfunction and greater anti-cancer potency. In an alternate embodiment,the antibodies of the present invention are optimized to have reduced orablated affinity for a human FcγR, including but not limited to FcγRI,FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. These embodiments areanticipated to provide antibodies with enhanced therapeutic propertiesin humans, for example reduced effector function and reduced toxicity.In other embodiments, antibodies of the present invention provideenhanced affinity for one or more FcγRs, yet reduced affinity for one ormore other FcγRs. For example, an antibody of the present invention mayhave enhanced binding to FcγRIIIa, yet reduced binding to FcγRIIb.Alternately, an antibody of the present invention may have enhancedbinding to FcγRIIa and FcγRI, yet reduced binding to FcγRIIb. In yetanother embodiment, an antibody of the present invention may haveenhanced affinity for FcγRIIb, yet reduced affinity to one or moreactivating FcγRs.

The modification of the invention preferably enhance binding affinityfor one or more FcγRs. By “greater affinity” or “improved affinity” or“enhanced affinity” or “better affinity” than a parent immunoglobulin,as used herein is meant that an Fc variant binds to an Fc receptor witha significantly higher equilibrium constant of association (KA) or lowerequilibrium constant of dissociation (KD) than the parent polypeptidewhen the amounts of variant and parent polypeptide in the binding assayare essentially the same. For example, the Fc variant with improved FcγRbinding affinity may display from about 5 fold to about 1000 fold, e.g.from about 10 fold to about 500 fold improvement in Fc receptor bindingaffinity compared to the parent polypeptide, where Fc receptor bindingaffinity is determined, for example, as disclosed in the Examplesherein. Accordingly, by “reduced affinity” as compared to a parent Fcpolypeptide as used herein is meant that an Fc variant binds an Fcreceptor with significantly lower KA or higher KD than the parentpolypeptide.

Data in the present study indicate that human WT IgG1 binds to humanV158 FcγRIIIa with an affinity of approximately 240 nM (Example 1). Thisis consistent with the literature which indicate that binding isapproximately 200-500 nM, as determined by Biacore (210 nM as shown inOkazaki et al, 2004, J Mol Bio 336:1239-49; 250 nM as shown in Lazar etal, Proc Natl Acad Sci USA 103(11):4005-4010) and calorimetry (530 nM,Okazaki et al, 2004, J Mol Bio 336:1239-49). However affinity as low as750 nM was measured in one study (Ferrara et al., 2006, J Biol Chem281(8):5032-5036). Although binding to F158 FcγRIIIa was lower than the5 uM cutoff applied in the present study, the literature indicates thathuman WT IgG1 binds to human F158 FcγRIIIa with an affinity ofapproximately 3-5 uM, as indicated by calorimetry (2.7 uM, in Okazaki etal, 2004, J Mol Bio 336:1239-49) and Biacore (5.0 uM, Ferrara et al.,2006, J Biol Chem 281(8):5032-5036).

Preferred embodiments comprise optimization of Fc binding to a humanFcγR, however in alternate embodiments the antibodies of the presentinvention possess enhanced or reduced affinity for FcγRs from nonhumanorganisms, including but not limited to rodents and non-human primates,antibodies that are optimized for binding to a nonhuman FcγR may finduse in experimentation. For example, mouse models are available for avariety of diseases that enable testing of properties such as efficacy,toxicity, and pharmacokinetics for a given drug candidate. As is knownin the art, cancer cells can be grafted or injected into mice to mimic ahuman cancer, a process referred to as xenografting. Testing ofantibodies that comprise antibodies that are optimized for one or moremouse FcγRs, may provide valuable information with regard to theefficacy of the protein, its mechanism of action, and the like. Theantibodies of the present invention may also be optimized for enhancedfunctionality and/or solution properties in aglycosylated form. In apreferred embodiment, the aglycosylated antibodies of the presentinvention bind an Fc ligand with greater affinity than the aglycosylatedform of the parent antibody. Said Fc ligands include but are not limitedto FcγRs, C1q, FcRn, and proteins A and G, and may be from any sourceincluding but not limited to human, mouse, rate, rabbit, or monkey,preferably human. In an alternately preferred embodiment, the antibodiesare optimized to be more stable and/or more soluble than theaglycosylated form of the parent antibody.

Antibodies of the invention may comprise modifications that modulateinteraction with Fc ligands other than FcγRs, including but not limitedto complement proteins, FcRn, and Fc receptor homologs (FcRHs). FcRHsinclude but are not limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, andFcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136, incorporatedentirely by reference).

Preferably, the Fc ligand specificity of the antibody of the presentinvention will determine its therapeutic utility. The utility of a givenantibody for therapeutic purposes will depend on the epitope or form ofthe target antigen and the disease or indication being treated. For sometargets and indications, enhanced FcγR-mediated effector functions maybe preferably. This may be particularly favorable for anti-cancerantibodies. Thus antibodies may be used that comprise antibodies thatprovide enhanced affinity for activating FcγRs and/or reduced affinityfor inhibitory FcγRs. For some targets and indications, it may befurther beneficial to utilize antibodies that provide differentialselectivity for different activating FcγRs; for example, in some casesenhanced binding to FcγRIIa and FcγRIIIa may be desired, but not FcγRI,whereas in other cases, enhanced binding only to FcγRIIa may bepreferred. For certain targets and indications, it may be preferable toutilize antibodies that enhance both FcγR-mediated andcomplement-mediated effector functions, whereas for other cases it maybe advantageous to utilize antibodies that enhance either FcγR-mediatedor complement-mediated effector functions. For some targets or cancerindications, it may be advantageous to reduce or ablate one or moreeffector functions, for example by knocking out binding to C1q, one ormore FcγR's, FcRn, or one or more other Fc ligands. For other targetsand indications, it may be preferable to utilize antibodies that provideenhanced binding to the inhibitory FcγRIIIb, yet WT level, reduced, orablated binding to activating FcγRs. This may be particularly useful,for example, when the goal of an antibody is to inhibit inflammation orauto-immune disease, or modulate the immune system in some way.

Clearly an important parameter that determines the most beneficialselectivity of a given antibody to treat a given disease is the contextof the antibody, that is what type of antibody is being used. Thus theFc ligand selectivity or specificity of a given antibody will providedifferent properties depending on whether it composes an antibody or anantibodies with a coupled fusion or conjugate partner. For example,toxin, radionucleotide, or other conjugates may be less toxic to normalcells if the antibody that comprises them has reduced or ablated bindingto one or more Fc ligands. As another example, in order to inhibitinflammation or auto-immune disease, it may be preferable to utilize anantibody with enhanced affinity for activating FcγRs, such as to bindthese FcγRs and prevent their activation. Conversely, an antibody thatcomprises two or more Fc regions with enhanced FcγRIIb affinity mayco-engage this receptor on the surface of immune cells, therebyinhibiting proliferation of these cells. Whereas in some cases anantibodies may engage its target antigen on one cell type yet engageFcγRs on separate cells from the target antigen, in other cases it maybe advantageous to engage FcγRs on the surface of the same cells as thetarget antigen. For example, if an antibody targets an antigen on a cellthat also expresses one or more FcγRs, it may be beneficial to utilizean antibody that enhances or reduces binding to the FcγRs on the surfaceof that cell. This may be the case, for example when the antibody isbeing used as an anti-cancer agent, and co-engagement of target antigenand FcγR on the surface of the same cell promote signaling events withinthe cell that result in growth inhibition, apoptosis, or otheranti-proliferative effect. Alternatively, antigen and FcγR co-engagementon the same cell may be advantageous when the antibody is being used tomodulate the immune system in some way, wherein co-engagement of targetantigen and FcγR provides some proliferative or anti-proliferativeeffect. Likewise, antibodies that comprise two or more Fc regions maybenefit from antibodies that modulate FcγR selectivity or specificity toco-engage FcγRs on the surface of the same cell.

The Fc ligand specificity of the antibodies of the present invention canbe modulated to create different effector function profiles that may besuited for particular antigen epitopes, indications or patientpopulations. FIG. 5 describes several preferred embodiments of receptorbinding profiles that include improvements to, reductions to or noeffect to the binding to various receptors, where such changes may bebeneficial in certain contexts. The receptor binding profiles in FIG. 5could be varied by degree of increase or decrease to the specifiedreceptors. Additionally, the binding changes specified could be in thecontext of additional binding changes to other receptors such as C1q orFcRn, for example by combining with ablation of binding to C1q to shutoff complement activation, or by combining with enhanced binding to C1qto increase complement activation. Other embodiments with other receptorbinding profiles are possible, the listed receptor binding profiles areexemplary.

The presence of different polymorphic forms of FcγRs provides yetanother parameter that impacts the therapeutic utility of the antibodiesof the present invention. Whereas the specificity and selectivity of agiven antibody for the different classes of FcγRs significantly affectsthe capacity of an antibody to target a given antigen for treatment of agiven disease, the specificity or selectivity of an antibody fordifferent polymorphic forms of these receptors may in part determinewhich research or pre-clinical experiments may be appropriate fortesting, and ultimately which patient populations may or may not respondto treatment. Thus the specificity or selectivity of antibodies of thepresent invention to Fc ligand polymorphisms, including but not limitedto FcγR, C1q, FcRn, and FcRH polymorphisms, may be used to guide theselection of valid research and pre-clinical experiments, clinical trialdesign, patient selection, dosing dependence, and/or other aspectsconcerning clinical trials.

Other Modifications

Antibodies of the present invention may comprise one or moremodifications that provide optimized properties that are notspecifically related to effector function per se. Said modifications maybe amino acid modifications, or may be modifications that are madeenzymatically or chemically. Such modification(s) likely provide someimprovement in the antibody, for example an enhancement in itsstability, solubility, function or clinical use. The present inventioncontemplates a variety of improvements that made be made by coupling theantibodies of the present invention with additional modifications.

In one embodiment, the variable region of an antibody of the presentinvention may be affinity matured, that is to say that amino acidmodifications have been made in the VH and/or VL domains of the antibodyto enhance binding of the antibody to its target antigen. Such types ofmodifications may improve the association and/or the dissociationkinetics for binding to the target antigen. Other modifications includethose that improve selectivity for target antigen vs. alternativetargets. These include modifications that improve selectivity forantigen expressed on target vs. non-target cells. Other improvements tothe target recognition properties may be provided by additionalmodifications. Such properties may include, but are not limited to,specific kinetic properties (i.e. association and dissociationkinetics), selectivity for the particular target versus alternativetargets, and selectivity for a specific form of target versusalternative forms. Examples include full-length versus splice variants,cell-surface vs. soluble forms, selectivity for various polymorphicvariants, or selectivity for specific conformational forms of the targetantigen.

Antibodies of the invention may comprise one or more modifications thatprovide reduced or enhanced internalization of an antibody. In oneembodiment, antibodies of the present invention can be utilized orcombined with additional modifications in order to reduce the cellularinternalization of an antibody that occurs via interaction with one ormore Fc ligands. This property might be expected to enhance effectorfunction, and potentially reduce immunogenicity of the antibodies of theinvention. Alternatively, antibodies of the present antibodies of thepresent invention can be utilized directly or combined with additionalmodifications in order to enhance the cellular internalization of anantibody that occurs via interaction with one or more Fc ligands. Forexample, in a preferred embodiment, an antibody is used that providesenhanced binding to FcγRI, which is expressed on dendritic cells andactive early in immune response. This strategy could be further enhancedby combination with additional modification, either within the antibodyor in an attached fusion or conjugate partner, that promote recognitionand presentation of FC peptide fragments by MHC molecules. Thesestrategies are expected to enhance target antigen processing and therebyimprove antigenicity of the target antigen (Bonnerot and Amigorena,1999, Immunol Rev. 172:279-84, incorporated entirely by reference),promoting an adaptive immune response and greater target cell killing bythe human immune system. These strategies may be particularlyadvantageous when the targeted antigen is shed from the cellularsurface. An additional application of these concepts arises withidiotype vaccine immunotherapies, in which clone-specific antibodiesproduced by a patient's lymphoma cells are used to vaccinate thepatient.

In a preferred embodiment, modifications are made to improve biophysicalproperties of the antibodies of the present invention, including but notlimited to stability, solubility, and oligomeric state. Modificationscan include, for example, substitutions that provide more favorableintramolecular interactions in the antibody such as to provide greaterstability, or substitution of exposed nonpolar amino acids with polaramino acids for higher solubility. A number of optimization goals andmethods are described in U.S. Ser. No. 10/379,392, incorporated entirelyby reference, that may find use for engineering additional modificationsto further optimize the antibodies of the present invention. Theantibodies of the present invention can also be combined with additionalmodifications that reduce oligomeric state or size, such that tumorpenetration is enhanced, or in vivo clearance rates are increased asdesired.

Other modifications to the antibodies of the present invention includethose that enable the specific formation or homodimeric orhomomultimeric molecules. Such modifications include but are not limitedto engineered disulfides, as well as chemical modifications oraggregation methods which may provide a mechanism for generatingcovalent homodimeric or homomultimers. For example, methods ofengineering and compositions of such molecules are described in Kan etal., 2001, J. Immunol., 2001, 166: 1320-1326; Stevenson et al., 2002,Recent Results Cancer Res. 159: 104-12; U.S. Pat. No. 5,681,566; Caronet al., 1992, J. Exp. Med. 176:1191-1195, and Shopes, 1992, J. Immunol.148(9):2918-22, all incorporated entirely by reference. Additionalmodifications to the variants of the present invention include thosethat enable the specific formation or heterodimeric, heteromultimeric,bifunctional, and/or multifunctional molecules. Such modificationsinclude, but are not limited to, one or more amino acid substitutions inthe CH3 domain, in which the substitutions reduce homodimer formationand increase heterodimer formation. For example, methods of engineeringand compositions of such molecules are described in Atwell et al., 1997,J. Mol. Biol. 270(1):26-35, and Carter et al., 2001, J. Immunol. Methods248:7-15, both incorporated entirely by reference. Additionalmodifications include modifications in the hinge and CH3 domains, inwhich the modifications reduce the propensity to form dimers.

In further embodiments, the antibodies of the present invention comprisemodifications that remove proteolytic degradation sites. These mayinclude, for example, protease sites that reduce production yields, aswell as protease sites that degrade the administered protein in vivo. Ina preferred embodiment, additional modifications are made to removecovalent degradation sites such as deamidation (i.e. deamidation ofglutaminyl and asparaginyl residues to the corresponding glutamyl andaspartyl residues), oxidation, and proteolytic degradation sites.Deamidation sites that are particular useful to remove are those thathave enhance propensity for deamidation, including, but not limited toasparaginyl and glutamyl residues followed by glycines (NG and QGmotifs, respectively). In such cases, substitution of either residue cansignificantly reduce the tendency for deamidation. Common oxidationsites include methionine and cysteine residues. Other covalentmodifications, that can either be introduced or removed, includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the “amino groups oflysine, arginine, and histidine side chains (T. E. Creighton, Proteins:Structure and Molecular Properties, W.H. Freeman & Co., San Francisco,pp. 79-86 (1983), incorporated entirely by reference), acetylation ofthe N-terminal amine, and amidation of any C-terminal carboxyl group.Additional modifications also may include but are not limited toposttranslational modifications such as N-linked or O-linkedglycosylation and phosphorylation.

Modifications may include those that improve expression and/orpurification yields from hosts or host cells commonly used forproduction of biologies. These include, but are not limited to variousmammalian cell lines (e.g. CHO), yeast cell lines, bacterial cell lines,and plants. Additional modifications include modifications that removeor reduce the ability of heavy chains to form inter-chain disulfidelinkages. Additional modifications include modifications that remove orreduce the ability of heavy chains to form intra-chain disulfidelinkages.

The antibodies of the present invention may comprise modification thatinclude the use of unnatural amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc. Natl. Acad. Sci.U.S.A. 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin etal., 2003, Science 301(5635):964-7, all incorporated entirely byreference. In some embodiments, these modifications enable manipulationof various functional, biophysical, immunological, or manufacturingproperties discussed above. In additional embodiments, thesemodifications enable additional chemical modification for otherpurposes. Other modifications are contemplated herein. For example, theantibody may be linked to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes,or copolymers of polyethylene glycol and polypropylene glycol.Additional amino acid modifications may be made to enable specific ornon-specific chemical or posttranslational modification of theantibodies. Such modifications, include, but are not limited toPEGylation and glycosylation. Specific substitutions that can beutilized to enable PEGylation include, but are not limited to,introduction of novel cysteine residues or unnatural amino acids suchthat efficient and specific coupling chemistries can be used to attach aPEG or otherwise polymeric moiety. Introduction of specificglycosylation sites can be achieved by introducing novel N-X-T/Ssequences into the antibodies of the present invention.

Covalent modifications of antibodies are included within the scope ofthis invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

In some embodiments, the covalent modification of the antibodies of theinvention comprises the addition of one or more labels. The term“labeling group” means any detectable label. In some embodiments, thelabeling group is coupled to the antibody via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and may be used in performing thepresent invention. In general, labels fall into a variety of classes,depending on the assay in which they are to be detected: a) isotopiclabels, which may be radioactive or heavy isotopes; b) magnetic labels(e.g. magnetic particles); c) redox active moieties; d) optical dyes;enzymatic groups (e.g. horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase); e) biotinylated groups; and f)predetermined polypeptide epitopes recognized by a secondary reporter(e.g., leucine sipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags, etc.). In someembodiments, the labeling group is coupled to the antibody via spacerarms of various lengths to reduce potential steric hindrance. Variousmethods for labeling proteins are known in the art and may be used inperforming the present invention. Specific labels include optical dyes,including, but not limited to, chromophores, phosphors and fluorophores,with the latter being specific in many instances. Fluorophores can beeither “small molecule” fluores, or proteinaceous fluores. By“fluorescent label” is meant any molecule that may be detected via itsinherent fluorescent properties.

Antibody Conjugates and Fusions

In one embodiment, the antibodies of the invention are antibody “fusionproteins”, sometimes referred to herein as “antibody conjugates”. Thefusion partner or conjugate partner can be proteinaceous ornon-proteinaceous; the latter generally being generated using functionalgroups on the antibody and on the conjugate partner. Conjugate andfusion partners may be any molecule, including small molecule chemicalcompounds and polypeptides. For example, a variety of antibodyconjugates and methods are described in Trail et al., 1990, Curr. Opin.Immunol. 11:584-588, incorporated entirely by reference. Possibleconjugate partners include but are not limited to cytokines, cytotoxicagents, toxins, radioisotopes, chemotherapeutic agent, anti-angiogenicagents, a tyrosine kinase inhibitors, and other therapeutically activeagents. In some embodiments, conjugate partners may be thought of moreas payloads, that is to say that the goal of a conjugate is targeteddelivery of the conjugate partner to a targeted cell, for example acaner cell or immune cell, by the antibody. Thus, for example, theconjugation of a toxin to an antibody targets the delivery of said toxinto cells expressing the target antigen. As will be appreciated by oneskilled in the art, in reality the concepts and definitions of fusionand conjugate are overlapping. The designation of an antibody as afusion or conjugate is not meant to constrain it in any particularembodiment of the present invention. Rather, these terms are usedloosely to convey the broad concept that any antibody of the presentinvention may be linked genetically, chemically, or otherwise, to one ormore polypeptides or molecules to provide some desirable property.

Suitable conjugates include, but are not limited to, labels as describedbelow, drugs and cytotoxic agents including, but not limited to,cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diptheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to antibodies, or binding of a radionuclide to a chelatingagent that has been covalently attached to the antibody. Additionalembodiments utilize calicheamicin, auristatins, geldanamycin,maytansine, and duocarmycins and analogs; for the latter, see U.S.2003/0050331, incorporated entirely by reference.

In one embodiment, the antibodies of the present invention are fused orconjugated to a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. For example, as described inPenichet et al., 2001, J. Immunol. Methods 248:91-101, incorporatedentirely by reference, cytokines may be fused to antibody to provide anarray of desirable properties. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormone such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, beta, and -gamma; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, a tumor necrosisfactor such as TNF-alpha or TNF-beta; C5a; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell culture;and biologically active equivalents of the native sequence cytokines.

In an alternate embodiment, the antibodies of the present invention arefused, conjugated, or operably linked to a toxin, including but notlimited to small molecule toxins and enzymatically active toxins ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof. For example, a variety of immunotoxins and immunotoxinmethods are described in Thrush et al., 1996, Ann. Rev. Immunol.14:49-71, incorporated entirely by reference. Small molecule toxinsinclude but are not limited to calicheamicin, maytansine (U.S. Pat. No.5,208,020, incorporated entirely by reference), trichothene, and CC1065.In one embodiment of the invention, the antibody is conjugated to one ormore maytansine molecules (e.g. about 1 to about 10 maytansine moleculesper antibody molecule). Maytansine may, for example, be converted toMay-SS-Mc which may be reduced to May-SH3 and reacted with modifiedantibody (Chari et al., 1992, Cancer Research 52: 127-131, incorporatedentirely by reference) to generate a maytansinoid-antibody conjugate.Another conjugate of interest comprises an antibody conjugated to one ormore calicheamicin molecules. The calicheamicin family of antibioticsare capable of producing double-stranded DNA breaks at sub-picomolarconcentrations. Structural analogues of calicheamicin that may be usedinclude but are not limited to γ₁ ¹, α₂ ¹, α₃, N-acetyl-γ₁ ¹, PSAG, andΘ¹ ₁, (Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al.,1998, Cancer Research 58:2925-2928) (U.S. Pat. No. 5,714,586; U.S. Pat.No. 5,712,374; U.S. Pat. No. 5,264,586; U.S. Pat. No. 5,773,001, allincorporated entirely by reference). Dolastatin 10 analogs such asauristatin E (AE) and monomethylauristatin E (MMAE) may find use asconjugates for the antibodies of the present invention (Doronina et al.,2003, Nat Biotechnol 21(7):778-84; Francisco et al., 2003 Blood102(4):1458-65, both incorporated entirely by reference). Usefulenzymatically active toxins include but are not limited to diphtheria Achain, nonbinding active fragments of diphtheria toxin, exotoxin A chain(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin Achain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordicacharantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor,gelonin, mitogellin, restrictocin, phenomycin, enomycin and thetricothecenes. See, for example, PCT WO 93/21232, incorporated entirelyby reference. The present invention further contemplates a conjugatebetween an antibody of the present invention and a compound withnucleolytic activity, for example a ribonuclease or DNA endonucleasesuch as a deoxyribonuclease (Dnase).

In an alternate embodiment, an antibody of the present invention may befused, conjugated, or operably linked to a radioisotope to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugate antibodies. Examples include, but are notlimited to At211, I131, I125, Y90, Re186, Sm153, Bi212, P32, andradioactive isotopes of Lu.

In yet another embodiment, an antibody of the present invention may beconjugated to a “receptor” (such streptavidin) for utilization in humorpretargeting wherein the antibody-receptor conjugate is administered tothe patient, followed by removal of unbound conjugate form thecirculation using a clearing agent and then administration of a “ligand”(e.g. avidin) which is conjugated to a cytotoxic agent (e.g. aradionucleotide). In an alternate embodiment, the antibody is conjugatedor operably linked to an enzyme in order to employ Antibody DependentEnzyme Mediated Prodrug Therapy (ADEPT). ADEPT may be used byconjugating or operably linking the antibody to a prodrug-activatingenzyme that converts a prodrug (e.g. a peptidyl chemotherapeutic agent,see PCT WO 81/01145, incorporated entirely by reference) to an activeanti-cancer drug. See, for example, PCT WO 88/07378 and U.S. Pat. No.4,975,278, both incorporated entirely by reference. The enzyme componentof the immunoconjugate useful for ADEPT includes any enzyme capable ofacting on a prodrug in such a way so as to convert it into its moreactive, cytotoxic form. Enzymes that are useful in the method of thisinvention include but are not limited to alkaline phosphatase useful forconverting phosphate-containing prodrugs into free drugs, arylsulfataseuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase useful for converting non-toxic 5-fluorocytosine intothe anti-cancer drug, 5-fluorouracil; proteases, such as serratia,protease, thermolysin, subtilisin, carboxypeptidases and cathepsins(such as cathepsins B and L), that are useful for convertingpeptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases,useful for converting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as .beta.-galactosidase andneuroamimidase useful for converting glycosylated prodrugs into freedrugs; beta-lactamase useful for converting drugs derivatized with.alpha.-lactams into free drugs; and penicillin amidases, such aspenicillin V amidase or penicillin G amidase, useful for convertingdrugs derivatized at their amine nitrogens with phenoxyacetyl orphenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, for example, Massey, 1987, Nature 328: 457-458, incorporatedherein entirely by reference). Antibody-abzyme conjugates can beprepared for delivery of the abzyme to a tumor cell population. Avariety of additional conjugates are contemplated for the antibodies ofthe present invention. A variety of chemotherapeutic agents,anti-angiogenic agents, tyrosine kinase inhibitors, and othertherapeutic agents are described below, which may find use as antibodyconjugates.

Also contemplated as fusion and conjugate partners are Fc polypeptides.Thus an antibody may be a multimer Fc polypeptide, comprising two ormore Fc regions. The advantage of such a molecule is that it providesmultiple binding sites for Fc receptors with a single protein molecule.In one embodiment, Fc regions may be linked using a chemical engineeringapproach. For example, Fab's and Fc's may be linked by thioether bondsoriginating at cysteine residues in the hinges, generating moleculessuch as FabFc₂. Fc regions may be linked using disulfide engineeringand/or chemical cross-linking. In a preferred embodiment, Fc regions maybe linked genetically. In a preferred embodiment, Fc regions in anantibody are linked genetically to generated tandemly linked Fc regionsas described in U.S. Ser. No. 11/022,289, filed Dec. 21, 2004, entitled“Fc polypeptides with novel Fc ligand binding sites,” incorporatedentirely by reference. Tandemly linked Fc polypeptides may comprise twoor more Fc regions, preferably one to three, most preferably two Fcregions. It may be advantageous to explore a number of engineeringconstructs in order to obtain homo- or hetero-tandemly linked antibodieswith the most favorable structural and functional properties. Tandemlylinked antibodies may be homo-tandemly linked antibodies, that is anantibody of one isotype is fused genetically to another antibody of thesame isotype. It is anticipated that because there are multiple Fc□R,C1q, and/or FcRn binding sites on tandemly linked Fc polypeptides,effector functions and/or pharmacokinetics may be enhanced. In analternate embodiment, antibodies from different isotypes may be tandemlylinked, referred to as hetero-tandemly linked antibodies. For example,because of the capacity to target FcγR and FCαRI receptors, an antibodythat binds both FcγRs and FcαRI may provide a significant clinicalimprovement.

In addition to antibodies, an antibody-like protein that is finding anexpanding role in research and therapy is the Fc fusion (Chamow et al.,1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr OpinImmunol 9:195-200, both incorporated entirely by reference). “Fc fusion”is herein meant to be synonymous with the terms “immunoadhesin”, “Igfusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes)as used in the prior art (Chamow et al., 1996, Trends Biotechnol14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200). An Fcfusion is a protein wherein one or more polypeptides is operably linkedto Fc. An Fc fusion combines the Fc region of an antibody, and thus itsfavorable effector functions and pharmacokinetics, with thetarget-binding region of a receptor, ligand, or some other protein orprotein domain. The role of the latter is to mediate target recognition,and thus it is functionally analogous to the antibody variable region.Because of the structural and functional overlap of Fc fusions withantibodies, the discussion on antibodies in the present inventionextends also to Fc.

Virtually any protein or small molecule may be linked to Fc to generatean Fc fusion. Protein fusion partners may include, but are not limitedto, the variable region of any antibody, the target-binding region of areceptor, an adhesion molecule, a ligand, an enzyme, a cytokine, achemokine, or some other protein or protein domain. Small moleculefusion partners may include any therapeutic agent that directs the Fcfusion to a therapeutic target. Such targets may be any molecule,preferably an extracellular receptor, that is implicated in disease.

Fusion and conjugates partners may be linked to any region of anantibody of the present invention, including at the N- or C-termini, orat some residue in between the termini. In a preferred embodiment, afusion or conjugate partner is linked at the N- or C-terminus of theantibody, most preferably the N-terminus. A variety of linkers may finduse in the present invention to covalently link antibodies to a fusionor conjugate partner. By “linker”, “linker sequence”, “spacer”,“tethering sequence” or grammatical equivalents thereof, herein is meanta molecule or group of molecules (such as a monomer or polymer) thatconnects two molecules and often serves to place the two molecules in apreferred configuration. Linkers are known in the art; for example,homo- or hetero-bifunctional linkers as are well known (see, 1994 PierceChemical Company catalog, technical section on cross-linkers, pages155-200, incorporated entirely by reference). A number of strategies maybe used to covalently link molecules together. These include, but arenot limited to polypeptide linkages between N- and C-termini of proteinsor protein domains, linkage via disulfide bonds, and linkage viachemical cross-linking reagents. In one aspect of this embodiment, thelinker is a peptide bond, generated by recombinant techniques or peptidesynthesis. The linker may contain amino acid residues that provideflexibility. Thus, the linker peptide may predominantly include thefollowing amino acid residues: Gly, Ser, Ala, or Thr. The linker peptideshould have a length that is adequate to link two molecules in such away that they assume the correct conformation relative to one another sothat they retain the desired activity. Suitable lengths for this purposeinclude at least one and not more than 50 amino acid residues.Preferably, the linker is from about 1 to 30 amino acids in length, withlinkers of 1 to 20 amino acids in length being most preferred. Usefullinkers include glycine-serine polymers (including, for example, (GS)n,(GSGGS)n (GGGGS)n and (GGGS)n, where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers, as will be appreciated by those in the art. Alternatively, avariety of nonproteinaceous polymers, including but not limited topolyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, orcopolymers of polyethylene glycol and polypropylene glycol, may find useas linkers, that is may find use to link the antibodies of the presentinvention to a fusion or conjugate partner, or to link the antibodies ofthe present invention to a conjugate.

Production of Antibodies

The present invention provides methods for producing and experimentallytesting antibodies. The described methods are not meant to constrain thepresent invention to any particular application or theory of operation.Rather, the provided methods are meant to illustrate generally that oneor more antibodies may be produced and experimentally tested to obtainvariant antibodies. General methods for antibody molecular biology,expression, purification, and screening are described in AntibodyEngineering, edited by Duebel & Kontermann, Springer-Verlag, Heidelberg,2001; and Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689;Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; Antibodies: ALaboratory Manual by Harlow & Lane, New York: Cold Spring HarborLaboratory Press, 1988, all incorporated entirely by reference.

In one embodiment of the present invention, nucleic acids are createdthat encode the antibodies, and that may then be cloned into host cells,expressed and assayed, if desired. Thus, nucleic acids, and particularlyDNA, may be made that encode each protein sequence. These practices arecarried out using well-known procedures. For example, a variety ofmethods that may find use in the present invention are described inMolecular Cloning—A Laboratory Manual, 3^(rd) Ed. (Maniatis, Cold SpringHarbor Laboratory Press, New York, 2001), and Current Protocols inMolecular Biology (John Wiley & Sons), both incorporated entirely byreference. As will be appreciated by those skilled in the art, thegeneration of exact sequences for a library comprising a large number ofsequences is potentially expensive and time consuming. By “library”herein is meant a set of variants in any form, including but not limitedto a list of nucleic acid or amino acid sequences, a list of nucleicacid or amino acid substitutions at variable positions, a physicallibrary comprising nucleic acids that encode the library sequences, or aphysical library comprising the variant proteins, either in purified orunpurified form. Accordingly, there are a variety of techniques that maybe used to efficiently generate libraries of the present invention. Suchmethods that may find use in the present invention are described orreferenced in U.S. Pat. No. 6,403,312; U.S. Ser. No. 09/782,004; U.S.Ser. No. 09/927,790; U.S. Ser. No. 10/218,102; PCT WO 01/40091; and PCTWO 02/25588, all incorporated entirely by reference. Such methodsinclude but are not limited to gene assembly methods, PCR-based methodand methods which use variations of PCR, ligase chain reaction-basedmethods, pooled oligo methods such as those used in synthetic shuffling,error-prone amplification methods and methods which use oligos withrandom mutations, classical site-directed mutagenesis methods, cassettemutagenesis, and other amplification and gene synthesis methods. As isknown in the art, there are a variety of commercially available kits andmethods for gene assembly, mutagenesis, vector subcloning, and the like,and such commercial products find use in the present invention forgenerating nucleic acids that encode antibodies.

The antibodies of the present invention may be produced by culturing ahost cell transformed with nucleic acid, preferably an expressionvector, containing nucleic acid encoding the antibodies, under theappropriate conditions to induce or cause expression of the protein. Theconditions appropriate for expression will vary with the choice of theexpression vector and the host cell, and will be easily ascertained byone skilled in the art through routine experimentation. A wide varietyof appropriate host cells may be used, including but not limited tomammalian cells, bacteria, insect cells, and yeast. For example, avariety of cell lines that may find use in the present invention aredescribed in the ATCC® cell line catalog, available from the AmericanType Culture Collection.

In a preferred embodiment, the antibodies are expressed in mammalianexpression systems, including systems in which the expression constructsare introduced into the mammalian cells using virus such as retrovirusor adenovirus. Any mammalian cells may be used, with human, mouse, rat,hamster, and primate cells being particularly preferred. Suitable cellsalso include known research cells, including but not limited to Jurkat Tcells, NIH3T3, CHO, BHK, COS, HEK293, PER C.6, HeLa, Sp2/0, NS0 cellsand variant thereof. In an alternatively preferred embodiment, libraryproteins are expressed in bacterial cells. Bacterial expression systemsare well known in the art, and include Escherichia coli (E. coli),Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans.In alternate embodiments, antibodies are produced in insect cells (e.g.Sf21/Sf9, Trichoplasia ni Bti-Tn5b1-4) or yeast cells (e.g. S.cerevisiae, Pichia, etc.). In an alternate embodiment, antibodies areexpressed in vitro using cell free insulation systems. In vitrotranslation systems derived from both prokaryotic (e.g. E. coli) andeukaryotic (e.g. wheat germ, rabbit reticulocytes) cells are availableand may be chosen based on the expression levels and functionalproperties of the protein of interest. For example, as appreciated bythose skilled in the art, in vitro translation is required for somedisplay technologies, for example ribosome display. In addition, theantibodies may be produced by chemical synthesis methods. Alsotransgenic expression systems both animal (e.g. cow, sheep or goat milk,embryonated hen's eggs, whole insect larvae, etc.) and plant (e.g. corn,tobacco, duckweed, etc.)

The nucleic acids that encode the antibodies of the present inventionmay be incorporated into an expression vector in order to express theprotein. A variety of expression vectors may be utilized for proteinexpression. Expression vectors may comprise self-replicatingextra-chromosomal vectors or vectors which integrate into a host genome.Expression vectors are constructed to be compatible with the host celltype. Thus expression vectors which find use in the present inventioninclude but are not limited to those which enable protein expression inmammalian cells, bacteria, insect cells, yeast, and in vitro systems. Asis known in the art, a variety of expression vectors are available,commercially or otherwise, that may find use in the present inventionfor expressing antibodies.

Expression vectors typically comprise a protein operably linked withcontrol or regulatory sequences, selectable markers, any fusionpartners, and/or additional elements. By “operably linked” herein ismeant that the nucleic acid is placed into a functional relationshipwith another nucleic acid sequence. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the antibody, and aretypically appropriate to the host cell need to express the protein. Ingeneral, the transcriptional and translational regulatory sequences mayinclude promoter sequences, ribosomal binding sites, transcriptionalstart and stop sequences, translational start and stop sequences, andenhancer or activator sequences. As is also known in the art, expressionvectors typically contain a selection gene or marker to allow theselection of transformed host cells containing the expression vector.Selection genes are well known in the art and will vary with the hostcell used.

Antibodies may be operably linked to a fusion partner to enabletargeting of the expressed protein, purification, screening, display,and the like. Fusion partners may be linked to the antibody sequence viaa linker sequences. The linker sequence will generally comprise a smallnumber of amino acids, typically less than ten, although longer linkersmay also be used. Typically, linker sequences are selected to beflexible and resistant to degradation. As will be appreciated by thoseskilled in the art, any of a wide variety of sequences may be used aslinkers. For example, a common linker sequence comprises the amino acidsequence GGGGS. A fusion partner may be a targeting or signal sequencethat directs antibody and any associated fusion partners to a desiredcellular location or to the extracellular media. As is known in the art,certain signaling sequences may target a protein to be either secretedinto the growth media, or into the periplasmic space, located betweenthe inner and outer membrane of the cell. A fusion partner may also be asequence that encodes a peptide or protein that enables purificationand/or screening. Such fusion partners include but are not limited topolyhistidine tags (His-tags) (for example H₆ and H₁₀ or other tags foruse with Immobilized Metal Affinity Chromatography (IMAC) systems (e.g.Ni⁺² affinity columns)), GST fusions, MBP fusions, Strep-tag, the BSPbiotinylation target sequence of the bacterial enzyme BirA, and epitopetags which are targeted by antibodies (for example o-myc tags,flag-tags, and the like). As will be appreciated by those skilled in theart, such tags may be useful for purification, for screening, or both.For example, an antibody may be purified using a His-tag by immobilizingit to a Ni⁺² affinity column, and then after purification the sameHis-tag may be used to immobilize the antibody to a Ni⁺² coated plate toperform an ELISA or other binding assay (as described below). A fusionpartner may enable the use of a selection method to screen antibodies(see below). Fusion partners that enable a variety of selection methodsare well-known in the art, and all of these find use in the presentinvention. For example, by fusing the members of an antibody library tothe gene III protein, phage display can be employed (Kay et al., Phagedisplay of peptides and proteins: a laboratory manual, Academic Press,Sand Diego, Calif., 1996; Lowman et al., 1991, Biochemistry30:10832-10838; Smith, 1998, Science 229:1315-1317, incorporatedentirely by reference). Fusion partners may enable antibodies to belabeled. Alternatively, a fusion partner may bind to a specific sequenceon the expression vector, enabling the fusion partner and associatedantibody to be linked covalently or noncovalently with the nucleic acidthat encodes them.

The methods of introducing exogenous nucleic acid into host cells arewell known in the art, and will vary with the host cell used. Techniquesinclude but are not limited to dextran-mediated transfection, calciumphosphate precipitation, calcium chloride treatment, polybrene mediatedtransfection, protoplast fusion, electroporation, viral or phageinfection, encapsulation of the polynucleotide(s) in liposomes, anddirect microinjection of the DNA into nuclei. In the case of mammaliancells, transfection may be either transient or stable.

In a preferred embodiment, antibodies are purified or isolated afterexpression. Proteins may be isolated or purified in a variety of waysknown to those skilled in the art. Standard purification methods includechromatographic techniques, including ion exchange, hydrophobicinteraction, affinity, sizing or gel filtration, and reversed-phase,carried out at atmospheric pressure or at high pressure using systemssuch as FPLC and HPLC. Purification methods also includeelectrophoretic, immunological, precipitation, dialysis, andchromatofocusing techniques. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.As is well known in the art, a variety of natural proteins bind Fc andantibodies, and these proteins can find use in the present invention forpurification of antibodies. For example, the bacterial proteins A and Gbind to the Fc region. Likewise, the bacterial protein L binds to theFab region of some antibodies, as of course does the antibody's targetantigen. Purification can often be enabled by a particular fusionpartner. For example, antibodies may be purified using glutathione resinif a GST fusion is employed, Ni⁺² affinity chromatography if a His-tagis employed, or immobilized anti-flag antibody if a flag-tag is used.For general guidance in suitable purification techniques, see, e.g.incorporated entirely by reference Protein Purification: Principles andPractice, 3^(rd) Ed., Scopes, Springer-Verlag, NY, 1994, incorporatedentirely by reference. The degree of purification necessary will varydepending on the screen or use of the antibodies. In some instances nopurification is necessary. For example in one embodiment, if theantibodies are secreted, screening may take place directly from themedia. As is well known in the art, some methods of selection do notinvolve purification of proteins. Thus, for example, if a library ofantibodies is made into a phage display library, protein purificationmay not be performed.

In Vitro Experimentation

Antibodies may be screened using a variety of methods, including but notlimited to those that use in vitro assays, in vivo and cell-basedassays, and selection technologies. Automation and high-throughputscreening technologies may be utilized in the screening procedures.Screening may employ the use of a fusion partner or label. The use offusion partners have discussed above. By “labeled” herein is meant thatthe antibodies of the invention have one or more elements, isotopes, orchemical compounds attached to enable the detection in a screen. Ingeneral, labels fall into three classes: a) immune labels, which may bean epitope incorporated as a fusion partner that is recognized by anantibody, b) isotopic labels, which may be radioactive or heavyisotopes, and c) small molecules labels, which may include fluorescentand colorimetric dyes, or molecules such as biotin that enable otherlabeling methods. Labels may be incorporated into the compound at anyposition and may be incorporated in vitro or in vivo during proteinexpression.

In a preferred embodiment, the function and/or biophysical properties ofantibodies are screened in an in vitro assay. In vitro assays may allowa broad dynamic range for screening properties of interest. Propertiesof antibodies that may be screened include but are not limited tostability, solubility, and affinity for Fc ligands, for example FcγRs.Multiple properties may be screened simultaneously or individually.Proteins may be purified or unpurified, depending on the requirements ofthe assay. In one embodiment, the screen is a qualitative orquantitative binding assay for binding of antibodies to a protein ornonprotein molecule that is known or thought to bind the antibody. In apreferred embodiment, the screen is a binding assay for measuringbinding to the target antigen. In an alternately preferred embodiment,the screen is an assay for binding of antibodies to an Fc ligand,including but are not limited to the family of FcγRs, the neonatalreceptor FcRn, the complement protein C1q, and the bacterial proteins Aand G. Said Fc ligands may be from any organism, with humans, mice,rats, rabbits, and monkeys preferred. Binding assays can be carried outusing a variety of methods known in the art, including but not limitedto FRET (Fluorescence Resonance Energy Transfer) and BRET(Bioluminescence Resonance Energy Transfer)-based assays, AlphaScreen™(Amplified Luminescent Proximity Homogeneous Assay), ScintillationProximity Assay, ELISA (Enzyme-Linked Immunosorbent Assay), SPR (SurfacePlasmon Resonance, also known as Biacore™), isothermal titrationcalorimetry, differential scanning calorimetry, gel electrophoresis, andchromatography including gel filtration. These and other methods maytake advantage of some fusion partner or label of the antibody. Assaysmay employ a variety of detection methods including but not limited tochromogenic, fluorescent, luminescent, or isotopic labels.

The biophysical properties of antibodies, for example stability andsolubility, may be screened using a variety of methods known in the art.Protein stability may be determined by measuring the thermodynamicequilibrium between folded and unfolded states. For example, antibodiesof the present invention may be unfolded using chemical denaturant,heat, or pH, and this transition may be monitored using methodsincluding but not limited to circular dichroism spectroscopy,fluorescence, spectroscopy, absorbance spectroscopy, NMR spectroscopy,calorimetry, and proteolysis. As will be appreciated by those skilled inthe art, the kinetic parameters of the folding and unfolding transitionsmay also be monitored using these and other techniques. The solubilityand overall structural integrity of an antibody may be quantitatively orqualitatively determined using a wide range of methods that are known inthe art. Methods which may find use in the present invention forcharacterizing the biophysical properties of antibodies include gelelectrophoresis, isoelectric focusing, capillary electrophoresis,chromatography such as size exclusion chromatography, ion-exchangechromatography, and reversed-phase high performance liquidchromatography, peptide mapping, oligosaccharide mapping, massspectrometry, ultraviolet absorbance spectroscopy, fluorescencespectroscopy, circular dichroism spectroscopy, isothermal titrationcalorimetry, differential scanning calorimetry, analyticultra-centrifugation, dynamic light scattering, proteolysis, andcross-linking, turbidity measurement, filter retardation assays,immunological assays, fluorescent dye binding assays, protein-stainingassays, microscopy, and detection of aggregates via ELISA or otherbinding assay. Structural analysis employing X-ray crystallographictechniques and NMR spectroscopy may also find use. In one embodiment,stability and/or solubility may be measured by determining the amount ofprotein solution after some defined period of time. In this assay, theprotein may or may not be exposed to some extreme condition, for exampleelevation temperature, low pH, or the presence of denaturant. Becausefunction typically requires a stable, soluble, and/orwell-folded/structured protein, the aforementioned functional andbinding assays also provide ways to perform such a measurement. Forexample, a solution comprising an antibody could be assayed for itsability to bind target antigen, then exposed to elevated temperature forone or more defined periods of time, then assayed for antigen bindingagain. Because unfolded and aggregated protein is not expected to becapable of binding antigen, the amount of activity remaining provides ameasure of the antibody's stability and solubility.

In a preferred embodiment, the library is screened using one or morecell-based or in vitro assays. For such assays, antibodies, purified orunpurified, are typically added exogenously such that cells are exposedto individual variants or groups of variants belonging to a library.These assays are typically, but not always, based on the biology of theability of the antibody to bind to antigen and mediate some biochemicalevent, for example effector functions like cellular lysis, phagocytosis,ligand/receptor binding inhibition, inhibition of growth and/orproliferation, apoptosis and the like. Such assays often involvemonitoring the response of cells to antibody, for example cell survival,cell death, cellular phagocytosis, cell lysis, change in cellularmorphology, or transcriptional activation such as cellular expression ofa natural gene or reporter gene. For example, such assays may measurethe ability of antibodies to elicit ADCC, ADCP, or CDC. For some assaysadditional cells or components, that is in addition to the target cells,may need to be added, for example serum complement, or effector cellssuch as peripheral blood monocytes (PBMCs), NK cells, macrophages, andthe like. Such additional cells may be from any organism, preferablyhumans, mice, rat, rabbit, and monkey. Crosslinked or monomericantibodies may cause apoptosis of certain cell lines expressing theantibody's target antigen, or they may mediate attack on target cells byimmune cells which have been added to the assay. Methods for monitoringcell death or viability are known in the art, and include the use ofdyes, fluorophores, immunochemical, cytochemical, and radioactivereagents. For example, caspase assays or annexin-flourconjugates mayenable apoptosis to be measured, and uptake or release of radioactivesubstrates (e.g. Chromium-51 release assays) or the metabolic reductionof fluorescent dyes such as alamar blue may enable cell growth,proliferation or activation to be monitored. In a preferred embodiment,the DELFIA® EuTDA-based cytotoxicity assay (Perkin Elmer, Mass.) isused. Alternatively, dead or damaged target cells may be monitored bymeasuring the release of one or more natural intracellular proteins, forexample lactate dehydrogenase. Transcriptional activation may also serveas a method for assaying function in cell-based assays. In this case,response may be monitored by assaying for natural genes or proteinswhich may be upregulated or down-regulated, for example the release ofcertain interleukins may be measured, or alternatively readout may bevia a luciferase or GFP-reporter construct. Cell-based assays may alsoinvolve the measure of morphological changes of cells as a response tothe presence of an antibody. Cell types for such assays may beprokaryotic or eukaryotic, and a variety of cell lines that are known inthe art may be employed. Alternatively, cell-based screens are performedusing cells that have been transformed or transfected with nucleic acidsencoding the antibodies.

In vitro assays include but are not limited to binding assays, ADCC,CDC, phagocytosis, cytotoxicity, proliferation, apoptosis, necrosis,cell cycle arrest, peroxide/ozone release, chemotaxis of effector cells,inhibition of such assays by reduced effector function antibodies;ranges of activities such as >100× improvement or >100× reduction,blends of receptor activation and the assay outcomes that are expectedfrom such receptor profiles.

In Vivo Experimentation

The biological properties of the antibodies of the present invention maybe characterized in cell, tissue, and whole organism experiments. As isknow in the art, drugs are often tested in animals, including but notlimited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in orderto measure a drug's efficacy for treatment against a disease or diseasemodel, or to measure a drug's pharmacokinetics, toxicity, and otherproperties. Said animals may be referred to as disease models. Withrespect to the antibodies of the present invention, a particularchallenge arises when using animal models to evaluate the potential forin-human efficacy of candidate polypeptides—this is due, at least inpart, to the fact that antibodies that have a specific effect on theaffinity for a human Fc receptor may not have a similar affinity effectwith the orthologous animal receptor. These problems can be furtherexacerbated by the inevitable ambiguities associated with correctassignment of true orthologues (Mechetina et al., Immunogenetics, 200254:463-468, incorporated entirely by reference), and the fact that someorthologues simply do not exist in the animal (e.g. humans possess anFcγRIIa whereas mice do not). Therapeutics are often tested in mice,including but not limited to nude mice, SCID mice, xenograft mice, andtransgenic mice (including knockins and knockouts). For example, anantibody of the present invention that is intended as an anti-cancertherapeutic may be tested in a mouse cancer model, for example axenograft mouse. In this method, a tumor or tumor cell line is graftedonto or injected into a mouse, and subsequently the mouse is treatedwith the therapeutic to determine the ability of the antibody to reduceor inhibit cancer growth and metastasis. An alternative approach is theuse of a SCID murine model in which immune-deficient mice are injectedwith human Periferal Blood Lymphocytes (PBLs), conferring asemi-functional and human immune system—with an appropriate array ofhuman FcRs—to the mice that have subsequently been injected withantibodies or Fc-polypeptides that target injected human tumor cells. Insuch a model, the Fc-polypeptides that target the desired antigen (suchas her2/neo on SkOV3 ovarian cancer cells) interact with human PBLswithin the mice to engage tumoricidal effector functions. Suchexperimentation may provide meaningful data for determination of thepotential of said antibody to be used as a therapeutic. Any organism,preferably mammals, may be used for testing. For example because oftheir genetic similarity to humans, monkeys can be suitable therapeuticmodels, and thus may be used to test the efficacy, toxicity,pharmacokinetics, or other property of the antibodies required forapproval as drugs, and thus of course these experiments arecontemplated. Thus the antibodies of the present invention may be testedin humans to determine their therapeutic efficacy, toxicity,pharmacokinetics, and/or other clinical properties.

The antibodies of the present invention may confer superior performanceon Fc-containing therapeutics in animal models or in humans. Thereceptor binding profiles of such antibodies, as described in thisspecification, may, for example, be selected to increase the potency ofcytotoxic drugs or to target specific effector functions or effectorcells to improve the selectivity of the drug's action. Further, receptorbinding profiles can be selected that may reduce some or all effectorfunctions thereby reducing the side-effects or toxicity of suchFc-containing drug. For example, an antibody with reduced binding toFcγRIIIa, FcγI and FcγRIIa can be selected to eliminate mostcell-mediated effector function, or an antibody with reduced binding toC1q may be selected to limit complement-mediated effector functions. Insome contexts, such effector functions are known to have potential toxiceffects, therefore eliminating them may increase the safety of theFc-bearing drug and such improved safety may be characterized in animalmodels. In some contexts, such effector functions are known to mediatethe desirable therapeutic activity, therefore enhancing them mayincrease the activity or potency of the Fc-bearing drug and suchimproved activity or potency may be characterized in animal models.

Optimized antibodies can be tested in a variety of orthotopic tumormodels. These clinically relevant animal models are important in thestudy of pathophysiology and therapy of aggressive cancers likepancreatic, prostate and breast cancer. Immune deprived mice including,but not limited to athymic nude or SCID mice are frequently used inscoring of local and systemic tumor spread from the site of intraorgan(e.g. pancreas, prostate or mammary gland) injection of human tumorcells or fragments of donor patients.

In preferred embodiments, antibodies of the present invention may beassessed for efficacy in clinically relevant animal models of varioushuman diseases. In many cases, relevant models include varioustransgenic animals for specific tumor antigens.

Relevant transgenic models such as those that express human Fc receptors(e.g., CD16 including the gamma chain, FcγRI, RIIa/b, and others) couldbe used to evaluate and test antibodies and Fc-fusions in theirefficacy. The evaluation of antibodies by the introduction of humangenes that directly or indirectly mediate effector function in mice orother rodents that may enable physiological studies of efficacy in tumortoxicity or other diseases such as autoimmune disorders and RA. Human Fcreceptors such as FcγRIIIa may possess polymorphisms such as that inposition 158 V or F which would further enable the introduction ofspecific and combinations of human polymorphisms into rodents. Thevarious studies involving polymorphism-specific FcRs are not limited tothis section, however, and encompasses all discussions and applicationsof FcRs in general as specified in throughout this applicationantibodies of the present invention may confer superior activity onFc-containing drugs in such transgenic models, in particular variantswith binding profiles optimized for human FcγRIIIa mediated activity mayshow superior activity in transgenic CD16 mice. Similar improvements inefficacy in mice transgenic for the other human Fc receptors, e.g.FcγRIIa, FcγRI, etc. may be observed for antibodies with bindingprofiles optimized for the respective receptors. Mice transgenic formultiple human receptors would show improved activity for antibodieswith binding profiles optimized for the corresponding multiplereceptors, for example as outlined in FIG. 5.

Because of the difficulties and ambiguities associated with using animalmodels to characterize the potential efficacy of candidate therapeuticantibodies in a human patient, some variant polypeptides of the presentinvention may find utility as proxies for assessing potential in-humanefficacy. Such proxy molecules would preferably mimic—in the animalsystem—the FcR and/or complement biology of a corresponding candidatehuman antibody. This mimicry is most likely to be manifested by relativeassociation affinities between specific antibodies and animal vs. humanreceptors. For example, if one were using a mouse model to assess thepotential in-human efficacy of an antibody that has enhanced affinityfor human FcγRIIIa, an appropriate proxy variant would have enhancedaffinity for mouse FcγRIII-2 (mouse CD16-2). Alternatively if one wereusing a mouse model to assess the potential in-human efficacy of anantibody that has reduced affinity for the inhibitory human FcγRIIb, anappropriate proxy variant would have reduced affinity for mouse FcγRII.It should also be noted that the proxy antibodies could be created inthe context of a human antibody, an animal antibody, or both.

In a preferred embodiment, the testing of antibodies may include studyof efficacy in primates (e.g. cynomolgus monkey model) to facilitate theevaluation of depletion of specific target cells harboring targetantigen. Additional primate models include but not limited to that ofthe rhesus monkey and Fc polypetides in therapeutic studies ofautoimmune, transplantation and cancer.

Toxicity studies are performed to determine the antibody or Fc-fusionrelated-effects that cannot be evaluated in standard pharmacologyprofile or occur only after repeated administration of the agent. Mosttoxicity tests are performed in two species—a rodent and a non-rodent—toensure that any unexpected adverse effects are not overlooked before newtherapeutic entities are introduced into man. In general, these modelsmay measure a variety of toxicities including genotoxicity, chronictoxicity, immunogenicity, reproductive/developmental toxicity andcarcinogenicity. Including within the aforementioned parameters arestandard measurement of food consumption, bodyweight, antibodyformation, clinical chemistry, and macro- and microscopic examination ofstandard organs/tissues (e.g. cardiotoxicity). Additional parameters ofmeasurement are injection site trauma and the measurement ofneutralizing antibodies, if an. Traditionally, monoclonal antibodytherapeutics, naked or conjugated are evaluated for cross-reactivitywith normal tissues, immunogenicity/antibody production, conjugate orlinker toxicity and “bystander” toxicity of radiolabeled species.Nonetheless, such studies may have to be individualized to addressspecific concerns and following the guidance set by ICH S6 (Safetystudies for biotechnological products also noted above). As such, thegeneral principles are that the products are sufficiently wellcharacterized and for which impurities/contaminants have been removed,that the test material is comparable throughout development, and GLPcompliance.

The pharmacokinetics (PK) of the antibodies of the invention can bestudied in a variety of animal systems, with the most relevant beingnon-human primates such as the cynomolgus, rhesus monkeys. Single orrepeated, i.v/s.c. administrations over a dose range of 6000-fold(0.05-300 mg/kg) can be evaluated for the half-life (days to weeks)using plasma concentration and clearance as well as volume ofdistribution as a steady state and level of systemic absorbance can bemeasured. Examples of such parameters of measurement generally includemaximum observed plasma concentration (Cmax), the time to reach Cmax(Tmax), the area under the plasma concentration-time curve from time 0to infinity [AUC(0-inf] and apparent elimination half-life (T1/2).Additional measured parameters could include compartmental analysis ofconcentration-time data obtained following i.v. administration andbioavailability. Examples of pharmacological/toxicological studies usingcynomolgus have been established for Rituxan and Zevalin in whichmonoclonal antibodies to CD20 are cross-reactive. Biodistribution,dosimetry (for radiolabeled antibodies), and PK studies can also be donein rodent models. Such studies would evaluate tolerance at all dosesadministered, toxicity to local tissues, preferential localization torodent xenograft animal models, depletion of target cells (e.g. CD20positive cells).

The antibodies of the present invention may confer superiorpharmacokinetics on Fc-containing therapeutics in animal systems or inhumans. For example, increased binding to FcRn may increase thehalf-life and exposure of the Fc-containing drug. Alternatively,decreased binding to FcRn may decrease the half-life and exposure of theFc-containing drug in cases where reduced exposure is favorable such aswhen such drug has side-effects.

It is known in the art that the array of Fc receptors is differentiallyexpressed on various immune cell types, as well as in different tissues.Differential tissue distribution of Fc receptors may ultimately have animpact on the pharmacodynamic (PD) and pharmacokinetic (PK) propertiesof antibodies of the present invention. Because antibodies of thepresentation have varying affinities for the array of Fc receptors,further screening of the polypeptides for PD and/or PK properties may beextremely useful for defining the optimal balance of PD, PK, andtherapeutic efficacy conferred by each candidate polypeptide.

Pharmacodynamic studies may include, but are not limited to, targetingspecific tumor cells or blocking signaling mechanisms, measuringdepletion of target antigen expressing cells or signals, etc. Theantibodies of the present invention may target particular effector cellpopulations and thereby direct Fc-containing drugs to recruit certainactivities to improve potency or to increase penetration into aparticularly favorable physiological compartment. For example,neutrophil activity and localization can be targeted by an antibody thatpreferentially targets FcγRIIIb. Such pharmacodynamic effects may bedemonstrated in animal models or in humans.

Clinical Use

The antibodies of the present invention may be used for varioustherapeutic purposes. As will be appreciated by those in the art, theantibodies of the present invention may be used for any therapeuticpurpose that uses antibodies and the like. In a preferred embodiment,the antibodies are administered to a patient to treat disordersincluding but not limited to cancer, autoimmune and inflammatorydiseases, and infectious diseases.

A “patient” for the purposes of the present invention includes bothhumans and other animals, preferably mammals and most preferably humans.Thus the antibodies of the present invention have both human therapy andveterinary applications. The term “treatment” or “treating” in thepresent invention is meant to include therapeutic treatment, as well asprophylactic, or suppressive measures for a disease or disorder. Thus,for example, successful administration of an antibody prior to onset ofthe disease results in treatment of the disease. As another example,successful administration of an optimized antibody after clinicalmanifestation of the disease to combat the symptoms of the diseasecomprises treatment of the disease. “Treatment” and “treating” alsoencompasses administration of an optimized antibody after the appearanceof the diseases in order to eradicate the disease. Successfuladministration of an agent after onset and after clinical symptoms havedeveloped, with possible abatement of clinical symptoms and perhapsamelioration of the diseases, comprises treatment of the disease. Those“in need of treatment” include mammals already having the disease ordisorder, as well as those prone to having the disease or disorder,including those in which the disease or disorder is to be prevented.

In one embodiment, an antibody of the present invention is administeredto a patient having a disease involving inappropriate expression of aprotein or other molecule. Within the scope of the present inventionthis meant to include diseases and disorders characterized by aberrantproteins, due for example to alterations in the amount of a proteinpresent, protein localization, posttranslational modification,conformational state, the presence of a mutant or pathogen protein, etc.Similarly, the disease or disorder may be characterized by alterationsmolecules including but not limited to polysaccharides and gangliosides.An overabundance may be due to any case, including but not limited tooverexpression at the molecular level, prolonged or accumulatedappearance at the site of action, or increased activity of proteinrelative to normal. Included within this definition are diseases anddisorders characterized by a reduction of a protein. This reduction maybe due to any cause, including but not limited to reduced expression atthe molecular level, shortened or reduced appearance at the site ofaction, mutant forms of a protein, or decreased activity of a proteinrelative to normal. Such an overabundance or reduction of a protein canbe measured relative to normal expression, appearance, or activity of aprotein, and said measurement may play an important role in thedevelopment and/or clinical testing of the antibodies of the presentinvention.

By “cancer” and “cancerous” herein refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma),neuroendocrine tumors, mesothelioma, schwanoma, meningioma,adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.

More particular examples of such cancers include hematologicmalignancies, such as non-Hodgkin's lymphomas (NHL), NHL cancers includebut are not limited to Burkitt's lymphoma (BL), small lymphocyticlymphoma/chronic lymphocytic leukemia (SLL/CLL), mantle cell lymphoma(MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLCL),marginal zone lymphoma (MZL), hairy cell leukemia (HCL) andlymphoplasmacytic leukemia (LPL), extranodal marginal zone B-celllymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginalzone B cell lymphoma, mediastinal large cell lymphoma, intravascularlarge cell lymphoma, primary effusion lymphoma, precursorB-lymphoblastic leukemia/lymphoma, precursor T- and NK-cells lymphoma(precursor T lymphoblastic lymphoma, blastic NK cell lymphoma), tumorsof the mature T and NK cells, including peripheral T-cell lymphoma andleukemia (PTL), adult T-cell leukemia/T-cell lymphomas and largegranular lymphocytic leukemia, T-cell chronic lymphocyticleukemia/prolymphocytic leukemia, T-cell large granular lymphocyticleukemia, aggressive NK-cell leukemia, extranodal T-/NK cell lymphoma,enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma,anaplastic large cell lymphoma (ALCL), angiocetric andangioimmunoblastic T-cell lymphoma, mycosis fungoides/Sézary syndrome,and cutaneous T-cell lymphoma (CTCL). Other cancers that may betreatable by the antibodies of the invention include but are not limitedto Hodgkin's lymphoma, tumors of lymphocyte precursor cells, includingB-cell acute lymphoblastic leukemia/lymphoma (B-ALL), and T-cell acutelymphoblastic leukemia/lymphoma (T-ALL), thymoma, Langerhans cellhistocytosis, multiple myeloma, myeloid neoplasias such as acutemyelogenous leukemias (AML), including AML with maturation, AML withoutdifferentiation, acute promyelocytic leukemia, acute myelomonocyticleukemia, and acute monocytic leukemias, myelodysplastic syndromes, andchronic myeloproliferative disorders (MDS), including chronicmyelogenous leukemia (CML). Other cancers that may be treatable by theantibodies of the invention include but are not limited to tumors of thecentral nervous system such as glioma, glioblastoma, neuroblastoma,astrocytoma, medulloblastoma, ependymoma, and retinoblastoma; solidtumors of the head and neck (eg. nasopharyngeal cancer, salivary glandcarcinoma, and esophagael cancer), lung (eg. small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung and squamouscarcinoma of the lung), digestive system (eg. gastric or stomach cancerincluding gastrointestinal cancer, cancer of the bile duct or biliarytract, colon cancer, rectal cancer, colorectal cancer, and analcarcinoma), reproductive system (eg. testicular, penile, or prostatecancer, uterine, vaginal, vulval, cervical ovarian, and endometrialcancer), skin (eg. melanoma, basal cell carcinoma, squamous cell cancer,actinic keratosis), liver (eg. liver cancer, hepatic carcinoma,hepatocellular cancer, and hepatoma), bone (eg. osteoelastoma, andosteolytic bone cancers) additional tissues and organs (eg. pancreaticcancer, bladder cancer, kidney or renal cancer, thyroid cancer, breastcancer, cancer of the peritoneum, and Kaposi's sarcoma), and tumors ofthe vascular system (eg. angiosarcoma and hemagiopericytoma).

Preferred oncology indications that may be treated by anti-CD19antibodies of the invention include but are not limited to allnon-Hodgkin's lymphomas (NHL), especially refractory/resistant NHL,chronic lymphocytic leukemia (CLL), B-cell acute lymphoblasticleukemia/lymphoma (B-ALL), and mantle cell lymphoma (MCL).

Autoimmunity results from a breakdown of self-tolerance involvinghumoral and/or cell-mediated immune mechanisms in. Among of theconsequences of failure in central and/or peripheral tolerance, aresurvival and activation of self-reactive B cells and T cells. Severalautoimmune disease are defined by excessive activation of both B and/orT lymphocytes. Activation of these cells requires in cooperation,antigen engagement and co-stimulatory signals from interactinglymphocytes. Antibody-mediated depletion, inhibition,anti-proliferation, and/or blockade of B cells are therapeuticapproaches for the treatment of autoimmune disease.

By “autoimmune diseases” herein include allogenic islet graft rejection,alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies(ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune myocarditis, autoimmuneneutropenia, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, autoimmune urticaria, Behcet's disease, bullouspermphigoid, cardiomyopathy, Castleman's syndrome, celiacspruce-dermatitis, chronic fatigue immune disfunction syndrome, chronicinflammatory demyelinating polyneuropathy, Churg-Strauss syndrome,cicatrical pemphigoid, CREST syndrome, cold agglutimin disease, Crohn'sdisease, dermatomyositis, discoid lupus, essential mixedcryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis,glomerulonephritis, Grave's disease, Guillain-Barre, Goodpasture'ssyndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis,hemophilia A, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, IgM polyneuropathies, immune mediatedthrombocytopenia, juvenile arthritis, Kawasaki's disease, lichenplantus, lupus erthematosis, Meniere's disease, mixed connective tissuedisease, multiple sclerosis (MS), type I diabetes mellitus, myastheniagravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychrondritis, polyglandular syndromes, polymyalgia rheomatics,polymyositis and dermatomyositis, primary agammaglobinulinemia, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, Reynauld'sphenomenon, Reiter's syndrome, rheumatoid arthritis (RA), sarcoidosis,scleroderma, Sjogren's syndrome, solid organ transplant rejection,stiff-man syndrome, systemic lupus erythematosus (SLE), takayasuarteritis, temporal arteristis/giant cell arteritis, thromboticthrombocytopenia purpura, ulcerative colitis, uveitis, vasculitides suchas dermatitis herpetiformis vasculitis, vitiligo, and Wegner'sgranulomatosis.

Preferred autoimmune indications that may be treated by anti-CD19antibodies of the invention include but are not limited to rheumatoidarthritis (RA), systemic lupus erythematosus (SLE or lupus), multiplesclerosis, Sjogren's syndrome, and idiopathic thrombocytopenia purpura(ITP).

By “inflammatory disorders” herein include acute respiratory distresssyndrome ARDS), acute septic arthritis, adjuvant arthritis, juvenileidiopathic arthritis, allergic encephalomyelitis, allergic rhinitis,allergic vasculitis, allergy, asthma, atherosclerosis, chronicinflammation due to chronic bacterial or viral infectionis, chronicobstructive pulmonary disease (COPD), coronary artery disease,encephalitis, inflammatory bowel disease, inflammatory osteolysis,inflammation associated with acute and delayed hypersensitivityreaction, inflammation associated with tumors, peripheral nerve injuryor demyelinating disease, inflammation associated with tissue traumasuch as burns and ischemia, inflammation due to meningitis, multipleorgan injury syndrome, pulmonary fibrosis, sepsis and septic shock,Stevens-Johnson syndrome, undifferentiated arthropy, andundifferentiated spondyloarthropathy.

By “infectious disease” herein include diseases caused by pathogens suchas viruses, bacteria, fungi, protozoa, and parasites. Infectiousdiseases may be caused by viruses including adenovirus, cytomegalovirus,dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C,herpes simplex type I, herpes simplex type II, human immunodeficiencyvirus (HIV), human papilloma virus (HPV), influenza, measles, mumps,papova virus, polio, respiratory syncytial virus, rinderpest,rhinovirus, rotavirus, rubella, SARS virus, smallpox, viral meningitis,and the like. Infectious diseases may also be caused by bacteriaincluding Bacillus antracis, Borrelia burgdorferi, Campylobacter jejuni,Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani,Diptheria, E. coli, Legionella, Helicobacter pylori, Mycobacteriumrickettsia, Mycoplasma nesisseria, Pertussis, Pseudomonas aeruginosa, S.pneumonia, Streptococcus, Staphylococcus, Vibria cholerae, Yersiniapestis, and the like. Infectious disease may also be caused by fungisuch as Aspergillus fumigatus, Blastomyces dermatitidis, Candidaalbicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasmacapsulatum, Penicillium marneffci, and the like. Infectious disease mayalso be caused by protozoa and parasites such as chlamydia, kokzidioa,leishmania, malaria, rickettsia, trypanosoma, and the like.

Furthermore, antibodies of the present invention may be used to preventor treat additional conditions including but not limited to heartconditions such as congestive heart failure (CHF), myocarditis and otherconditions of the myocardium; skin conditions such as rosecea, acne, andeczema; bone and tooth conditions such as bone loss, osteoporosis,Paget's disease, Langerhans' cell histiocytosis, periodontal disease,disuse osteopenia, osteomalacia, monostotic fibrous dysplasia,polyostotic fibrous dysplasia, bone metastasis, bone pain management,humoral malignant hypercalcemia, periodontal reconstruction, spinal cordinjury, and bone fractures; metabolic conditions such as Gaucher'sdisease; endocrine conditions such as Cushing's syndrome; andneurological conditions.

A number of the receptors that may interact with the antibodies of thepresent invention are polymorphic in the human population. For a givenpatient or population of patients, the efficacy of the antibodies of thepresent invention may be affected by the presence or absence of specificpolymorphisms in proteins. For example, FcγRIIIA is polymorphic atposition 158, which is commonly either V (high affinity) or F (lowaffinity). Patients with the V/V homozygous genotype are observed tohave a better clinical response to treatment with the anti-CD20 antibodyRituxan® (rituximab), likely because these patients mount a stronger NKresponse (Dall'Ozzo et al. (2004) Cancer Res. 64:4664-9, incorporatedentirely by reference). Additional polymorphisms include but are notlimited to FcγRIIIA R131 or H131, and such polymorphisms are known toeither increase or decrease Fc binding and subsequent biologicalactivity, depending on the polymorphism, antibodies of the presentinvention may bind preferentially to a particular polymorphic form of areceptor, for example FcγRIIIA 158 V, or to bind with equivalentaffinity to all of the polymorphisms at a particular position in thereceptor, for example both the 158V and 158F polymorphisms of FcγRIIIA.In a preferred embodiment, antibodies of the present invention may haveequivalent binding to polymorphisms may be used in an antibody toeliminate the differential efficacy seen in patients with differentpolymorphisms. Such a property may give greater consistency intherapeutic response and reduce non-responding patient populations. Suchvariant Fc with identical binding to receptor polymorphisms may haveincreased biological activity, such as ADCC, CDC or circulatinghalf-life, or alternatively decreased activity, via modulation of thebinding to the relevant Fc receptors. In a preferred embodiment,antibodies of the present invention may bind with higher or loweraffinity to one of the polymorphisms of a receptor, either accentuatingthe existing difference in binding or reversing the difference. Such aproperty may allow creation of therapeutics particularly tailored forefficacy with a patient population possessing such polymorphism. Forexample, a patient population possessing a polymorphism with a higheraffinity for an inhibitory receptor such as FcγRIIB could receive a drugcontaining an antibody with reduced binding to such polymorphic form ofthe receptor, creating a more efficacious drug.

In a preferred embodiment, patients are screened for one or morepolymorphisms in order to predict the efficacy of the antibodies of thepresent invention. This information may be used, for example, to selectpatients to include or exclude from clinical trials or, post-approval,to provide guidance to physicians and patients regarding appropriatedosages and treatment options. For example, in patients that arehomozygous or heterozygous for FcγRIIIA 158F antibody drugs such as theanti-CD20 mAb, Rituximab are minimally effective (Carton 2002 Blood 99:754-758; Weng 2003 J. Clin. Oncol. 21:3940-3947, both incorporatedentirely by reference); such patients may show a much better clinicalresponse to the antibodies of the present invention. In one embodiment,patients are selected for inclusion in clinical trials for an antibodyof the present invention if their genotype indicates that they arelikely to respond significantly better to an antibody of the presentinvention as compared to one or more currently used antibodytherapeutics. In another embodiment, appropriate dosages and treatmentregimens are determined using such genotype information. In anotherembodiment, patients are selected for inclusion in a clinical trial orfor receipt of therapy post-approval based on their polymorphismgenotype, where such therapy contains an antibody engineered to bespecifically efficacious for such population, or alternatively wheresuch therapy contains an antibody that does not show differentialactivity to the different forms of the polymorphism.

Included in the present invention are diagnostic tests to identifypatients who are likely to show a favorable clinical response to anantibody of the present invention, or who are likely to exhibit asignificantly better response when treated with an antibody of thepresent invention versus one or more currently used antibodytherapeutics. Any of a number of methods for determining FcγRpolymorphisms in humans known in the art may be used.

Furthermore, the present invention comprises prognostic tests performedon clinical samples such as blood and tissue samples. Such tests mayassay for effector function activity, including but not limited to ADCC,CDC, phagocytosis, and opsonization, or for killing, regardless ofmechanism, of cancerous or otherwise pathogenic cells. In a preferredembodiment, ADCC assays, such as those described previously, are used topredict, for a specific patient, the efficacy of a given antibody of thepresent invention. Such information may be used to identify patients forinclusion or exclusion in clinical trials, or to inform decisionsregarding appropriate dosages and treatment regemins. Such informationmay also be used to select a drug that contains a particular antibodythat shows superior activity in such assay.

Formulation

Pharmaceutical compositions are contemplated wherein an antibody of thepresent invention and one or more therapeutically active agents areformulated. Formulations of the antibodies of the present invention areprepared for storage by mixing said antibody having the desired degreeof purity with optional pharmaceutically acceptable carriers, excipientsor stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed., 1980, incorporated entirely by reference), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,acetate, and other organic acids, antioxidants including ascorbic acidand methionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzothoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners andother flavoring agents; fillers such as microcrystalline cellulose,lactose, corn and other starches; binding agents; additives; coloringagents; salt-forming counter-ions such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG). In a preferred embodiment, thepharmaceutical composition that comprises the antibody of the presentinvention may be in a water-soluble form, such as being present aspharmaceutically acceptable salts, which is meant to include both acidand base addition salts. “Pharmaceutically acceptable acid additionsalt” refers to those salts that retain the biological effectiveness ofthe free bases and that are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theimmonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine. The formulations to beused for in vivo administration are preferably sterile. This is readilyaccomplished by filtration through sterile filtration membranes or othermethods.

The antibodies disclosed herein may also be formulated asimmunoliposomes. A liposome is a small vesicle comprising various typesof lipids, phospholipids and/or surfactant that is useful for deliveryof a therapeutic agent to a mammal. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., 1985, Proc Natl Acad Sci USA 82:3688; Hwang et al., 1980, ProcNatl Acad Sci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat. No.4,544,545; and PCT WO 97/38731, all incorporated entirely by reference.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556, incorporated entirely by reference. The components of theliposome are commonly arranged in a bilayer formation, similar to thelipid arrangement of biological membranes. Particularly useful liposomescan be generated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter. A chemotherapeutic agent or other therapeuticallyactive agent is optionally contained within the liposome (Gabizon etal., 1989, J National Cancer Inst 81:1484, incorporated entirely byreference).

The antibody and other therapeutically active agents may also beentrapped in microcapsules prepared by methods including but not limitedto coacervation techniques, interfacial polymerization (for exampleusing hydroxymethylcellulose or gelatin-microcapsules, orpoly-(methylmethacrylate) microcapsules), colloidal drug deliverysystems (for example, liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules), and macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed., 1980, incorporated entirely by reference. Sustained-releasepreparations may be prepared. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymer, which matrices are in the form of shaped articles, e.g. films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol), polylactides (U.S. Pat. No. 3,773,919, incorporatedentirely by reference), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LupronDepot® (which are injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate),poly-D-(−)-3-hydroxybutyric acid, and ProLease® (commercially availablefrom Alkermes), which is a microsphere-based delivery system composed ofthe desired bioactive molecule incorporated into a matrix ofpoly-DL-lactide-co-glycolide (PLG).

Administration

Administration of the pharmaceutical composition comprising an antibodyof the present invention, preferably in the form of a sterile aqueoussolution, may be done in a variety of ways, including, but not limitedto orally, subcutaneously, intravenously, intranasal, intraotically,transdermally, topically (e.g., gels, salves, lotions, creams, etc.),intraperitoneally, intramuscularly, intrapulmonary, vaginally,parenterally, rectally, or intraocularly. In some instances, for examplefor the treatment of wounds, inflammation, etc., the antibody may bedirectly applied as a solution or spray. As is known in the art, thepharmaceutical composition may be formulated accordingly depending uponthe manner of introduction.

Subcutaneous administration may be preferable in some circumstancesbecause the patient may self-administer the pharmaceutical composition.Many protein therapeutics are not sufficiently potent to allow forformulation of a therapeutically effective does in the maximumacceptable volume for subcutaneous administration. This problem may beaddress in part by the use of protein formulations comprisingarginine-HCl, histidine, and polysorbate (see WO 04091658, incorporatedentirely by reference). Antibodies of the present invention may be moreamenable to subcutaneous administration due to, for example, increasedpotency, improved serum half-life, or enhanced solubility.

As is known in the art, protein therapeutics are often delivered by IVinfusion or bolus. The antibodies of the present invention may also bedelivered using such methods. For example, administration may venious beby intravenous infusion with 0.9% sodium chloride as an infusionvehicle.

Pulmonary delivery may be accomplished using an inhaler or nebulizer anda formulation comprising an aerosolizing agent. For example, AERx®inhalable technology commercially available from Aradigm, or Inhance™pulmonary delivery system commercially available from NektarTherapeutics may be used. Antibodies of the present invention may bemore amenable to intrapulmonary delivery. FcRn is present in the lung,and may promote transport from the lung to the bloodstream (e.g.Syntonix WO 04004798, Bitonti et al. (2004) Proc. Nat. Acad. Sci.101:9763-8, both incorporated entirely by reference). Accordingly,antibodies that bind FcRn more effectively in the lung or that arereleased more efficiently in the bloodstream may have improvedbioavailability following intrapulmonary administration. Antibodies ofthe present invention may also be more amenable to intrapulmonaryadministration due to, for example, improved solubility or alteredisoelectric point.

Furthermore, antibodies of the present invention may be more amenable tooral delivery due to, for example, improved stability at gastric pH andincreased resistance to proteolysis. Furthermore, FcRn appears to beexpressed in the intestinal epithelia of adults (Dickinson et al. (1999)J. Clin. Invest. 104:903-11, incorporated entirely by reference), soantibodies of the present invention with improved FcRn interactionprofiles may show enhanced bioavailability following oraladministration. FcRn mediated transport of antibodies may also occur atother mucus membranes such as those in the gastrointestinal,respiratory, and genital trades (Yoshida et al. (2004) Immunity20:769-83, incorporated entirely by reference).

In addition, any of a number of delivery systems are known in the artand may be used to administer the antibodies of the present invention.Examples include, but are not limited to, encapsulation in liposomes,microparticles, microspheres (eg. PLA/PGA microspheres), and the like.Alternatively, an implant of a porous, non-porous, or gelatinousmaterial, including membranes or fibers, may be used. Sustained releasesystems may comprise a polymer material or matrix such as polyesters,hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamicacid and ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-glycolicacid copolymers such as the Lupron Depot®, andpoly-D-(−)-3-hydoxyburyric acid. It is also possible to administer anucleic acid encoding the antibody of the current invention, forexample, by retroviral infection, direct injection, or coating withlipids, cell surface receptors, or other transfection agents. In allcases, controlled release systems may be used to release the antibody ator close to the desired location of action.

Dosing

The dosing amounts and frequencies of administration are, in a preferredembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

The concentration of the therapeutically active antibody in theformulation may vary from about 0.1 to 100 weight %. In a preferredembodiment, the concentration of the antibody is in the range of 0.003to 1.0 molar. In order to treat a patient, a therapeutically effectivedose of the antibody of the present invention may be administered. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. Dosages may range from 0.0001 to 100mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg ofbody weight, with 1 to 10 mg/kg being preferred.

In some embodiments, only a single dose of the antibody is used. Inother embodiments, multiple doses of the antibody are administered. Theelapsed time between administration may be less than 1 hour, about 1hour, about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6 hours,about 12 hours, about 24 hours, about 48 hours, about 2-4 days, about4-6 days, about 1 week, about 2 weeks, or more than 2 weeks.

In other embodiments the antibodies of the present invention areadministered in metronomic dosing regimes, either by continuous infusionor frequent administration without extended rest periods. Suchmetronomic administration may involve dosing at constant intervalswithout rest periods. Typically such regimens encompass chronic low-doseor continuous infusion for an extended period of time, for example 1-2days, 1-2 weeks, 1-2 months, or up to 6 months or more. The use of lowerdoses may minimize side effects and the need for rest periods.

In certain embodiments the antibody of the present invention and one ormore other prophylactic or therapeutic agents are cyclicallyadministered to the patient. Cycling therapy involves administration ofa first agent at one time, a second agent at a second time, optionallyadditional agents at additional times, optionally a rest period, andthen repeating this sequence of administration one or more times. Thenumber of cycles is typically from 2-10. Cycling therapy may reduce thedevelopment of resistance to one or more agents, may minimize sideeffects, or may improve treatment efficacy.

Combination Therapies

The antibodies of the present invention may be administeredconcomitantly with one or more other therapeutic regimens or agents. Theadditional therapeutic regimes or agents may be used to improve theefficacy or safety of the antibody. Also, the additional therapeuticregimes or agents may be used to treat the same disease or a comorbidityrather than to alter the action of the antibody. For example, anantibody of the present invention may be administered to the patientalong with chemotherapy, radiation therapy, or both chemotherapy andradiation therapy. The antibody of the present invention may beadministered in combination with one or more other prophylactic ortherapeutic agents, including but not limited to cytotoxic agents,chemotherapeutic agents, cytokines, growth inhibitory agents,anti-hormonal agents, kinase inhibitors, anti-angiogenic agents,cardioprotectants, immunostimulatory agents, immunosuppressive agents,agents that promote proliferation of hematological cells, angiogenesisinhibitors, protein tyrosine kinase (PTK) inhibitors, additionalantibodies, FcγRIIb or other Fc receptor inhibitors, or othertherapeutic agents.

The terms “in combination with” and “co-administration” are not limitedto the administration of said prophylactic or therapeutic agents atexactly the same time. Instead, it is meant that the antibody of thepresent invention and the other agent or agents are administered in asequence and within a time interval such that they may act together toprovide a benefit that is increased versus treatment with only eitherthe antibody of the present invention or the other agent or agents. Itis preferred that the antibody and the other agent or agents actadditively, and especially preferred that they act synergistically. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended. The skilled medical practitioner candetermine empirically, or by considering the pharmacokinetics and modesof action of the agents, the appropriate dose or doses of eachtherapeutic agent, as well as the appropriate timings and methods ofadministration.

In one embodiment, the antibodies of the present invention areadministered with one or more additional molecules comprising antibodiesor Fc. The antibodies of the present invention may be co-administeredwith one or more other antibodies that have efficacy in treating thesame disease or an additional comorbidity, for example two antibodiesmay be administered that recognize two antigens that are overexpressedin a given type of cancer, or two antigens that mediate pathogenesis ofan autoimmune or infectious disease.

Examples of anti-cancer antibodies that may be co-administered include,but are not limited to, anti-17-1A cell surface antigen antibodies suchas Panorex™ (edrecolomab); anti-4-1BB antibodies; anti-4Dc antibodies;anti-A33 antibodies such as A33 and CDP-833; anti-α4β1 integrinantibodies such as natalizumab; anti-α4β7 integrin antibodies such asLDP-02; anti-αVβ1 integrin antibodies such as F-200, M-200, and SJ-749;anti-αVβ3 integrin antibodies such as adciximab, CNTO-95, Mab-17E6, andVitaxin™; anti-complement factor 5 (C5) antibodies such as 5G1.1;anti-CA125 antibodies such as OvaRex® (oregovomab); anti-CD3 antibodiessuch as Nuvion® (visilizumab) and Rexomab; anti-CD4 antibodies such asIDEC-151, MDX-CD4, OKT4A, anti-CD6 antibodies such as Oncolysin B andOncolysin CD6; anti-CD7 antibodies such as HB2; anti-CD19 antibodiessuch as B43, MT-103, and Oncolysin B; anti-CD20 antibodies such as 2H7,2H7.v16, 2H7.v114, 2H7.v115, Bexxar® (tositumomab, I-131 labeledanti-CD20); Ritoxan® (rituximab), and Zevalin® (Ibritumomab tiuxetan,Y-90 labeled anti-CD20); anti-CD22 antibodies such as Lymphocide™(epratuzumab, Y-90 labeled anti-CD22); anti-CD23 antibodies such asIDEC-152; anti-CD25 antibodies such as basiliximab and Zenapax®(daclizumab); anti-CD30 antibodies such as AC10, MDX-060, and SGN-30;anti-CD33 antibodies such as Mylotarg® (gemtazumab ozogamicin),Oncolysin M, and Smart M195; anti-CD38 antibodies; anti-CD40 antibodiessuch as SGN-40 and toralizumab; anti-CD40 antibodies such as 5c8,Antova™, and IDEC-131; anti-CD44 antibodies such as bivatuzumab;anti-CD46 antibodies, anti-CD52 antibodies such as Campath®(alemtuzumab); anti-CD55 antibodies such as SC-1; anti-CD56 antibodiessuch as huN901-DM1; anti-CD64 antibodies such as MDX-33; anti-CD66eantibodies such as XR-303; anti-CD74 antibodies such as IMMU-110;anti-CD80 antibodies such as galiximab and IDEC-114; anti-CD89antibodies such as MDX-214; anti-CD123 antibodies; anti-CD138 antibodiessuch as B-B4-DM1; anti-CD146 antibodies such as AA-98; anti-CD148antibodies; anti-CEA antibodies such as cT84.66, labetuzumab, andPentacca™; anti-CTLA-4 antibodies such as MDX-101; anti-CXCR4antibodies; anti-EGFR antibodies such as ABX-EGF, Erbitux® (cetuximab),IMC-C225, and Merck Mab 425; anti-EpCAM antibodies such as Crucell'santi-EpCAM, ING-1, and IS-IL-2; anti-ephrin B2/EphB4 antibodies;anti-Her2 antibodies such as Herceptin®, MDX-210; anti-FAP (fibroblastactivation protein) antibodies such as sibrotuzumab; anti-ferritinantibodies such as NXT-211; anti-FGF-1 antibodies; anti-FGF-3antibodies; anti-FGF-8 antibodies; anti-FGFR antibodies, anti-fibrinantibodies; anti-G250 antibodies such as WX-G250 and Rencarex®; anti-GD2ganglioside antibodies such as EMD-273063 and TriGem; anti-GD3ganglioside antibodies such as BEC2, KW-2871, and mitumomab;anti-gpIIb/IIIa antibodies such as ReoPro; anti-heparinase antibodies;anti-Her2/ErbB2 antibodies such as Herceptin® (trastuzumab), MDX-210,and pertuzumab; anti-HLA antibodies such as Oncolym®, Smart ID10;anti-HM1.24 antibodies; anti-ICAM antibodies such as ICM3; anti-IgAreceptor antibodies; anti-IGF-1 antibodies such as CP-751871 and EM-164;anti-IGF-1R antibodies such as IMC-A12; anti-IL-6 antibodies such asCNTO-328 and elsilimomab; anti-IL-15 antibodies such as HuMax™-IL15;anti-KDR antibodies; anti-laminin 5 antibodies, anti-Lewis Y antigenantibodies such as Hu38193 and IGN-311; anti-MCAM antibodies; anti-Muc1antibodies such as BravaRex and TriAb; anti-NCAM antibodies such asERIC-1 and ICRT; anti-PEM antigen antibodies such as Theragyn andTherex; anti-PSA antibodies; anti-PSCA antibodies such as IO8; anti-Ptkantibodies; anti-PTN antibodies; anti-RANKI, antibodies such as AMG-162;anti-RLIP76 antibodies; anti-SK-1 antigen antibodies such as MonopharmC; anti-STEAP antibodies; anti-TAG72 antibodies such as CC49-SCA andMDX-220; anti-TGF-β antibodies such as CAT-152; anti-TNF-α antibodiessuch as CDP571, CDP870, D2E7, Humira® (adalimumab), and Remicade®(infliximab); anti-TRAIL-R1 and TRAIL-R2 antibodies; anti-VE-cadherin-2antibodies; and anti-VLA-4 antibodies such as Antegren™. Furthermore,anti-idiotype antibodies including but not limited to the CD3 epitopeantibody BEC2 and the gp72 epitope antibody 105AD7, may be used. Inaddition, bispecific antibodies including but not limited to theanti-CD3/CD20 antibody Bi20 may be used.

Examples of antibodies that may be co-administered to treat autoimmuneor inflammatory disease, transplant rejection, GVHD, and the likeinclude, but are not limited to, anti-α4β7 integrin antibodies such asLDP-02, anti-beta2 integrin antibodies such as LDP-01, anti-complement(C5) antibodies such as 5G1.1, anti-CD2 antibodies such as BTI-322,MEDI-507, anti-CD3 antibodies such as OKT3, SMART anti-CD3, anti-CD4antibodies such as IDEC-151, MDX-CD4, OKT4A, anti-CD11a antibodies,anti-CD14 antibodies such as ICI4, anti-CD18 antibodies, anti-CD23antibodies such as IDEC 152, anti-CD25 antibodies such as Zenapax,anti-CD40L antibodies such as 5c8, Antova, IDEC-131, anti-CD64antibodies such as MDX-33, anti-CD80 antibodies such as IDEC-114,anti-CD147 antibodies such as ABX-CBL, anti-E-selectin antibodies suchas CDP850, anti-gpIIb/IIIa antibodies such as ReoPro/Abcixima,anti-ICAM-3 antibodies such as ICM3, anti-ICE antibodies such as VX-740,anti-FcγRI antibodies such as MDX-33, anti-IgE antibodies such asrhuMab-E25, anti-IL-4 antibodies such as SB-240683, anti-IL-5 antibodiessuch as SB-240563, SCH55700, anti-IL-8 antibodies such as ABX-IL8,anti-interferon gamma antibodies, and anti-TNFa antibodies such asCDP571, CDP870, D2E7, Infliximab, MAK-195F, anti-VLA-4 antibodies suchas Antegren. Examples of other Fc-containing molecules that may beco-administered to treat autoimmune or inflammatory disease, transplantrejection, GVHD, and the like include, but are not limited to, the p75TNF receptor/Fc fusion Enbrel® (etanercept) and Regeneron's IL-1 trap.

Examples of antibodies that may be co-administered to treat infectiousdiseases include, but are not limited to, anti-anthrax antibodies suchas ABthrax, anti-CMV antibodies such as CytoGam and sevirumab,anti-cryptosporidium antibodies such as CryptoGAM, Sporidin-G,anti-helicobacter antibodies such as Pyloran, anti-hepatitis Bantibodies such as HepeX-B, Nabi-HB, anti-HIV antibodies such asHRG-214, anti-RSV antibodies such as felvizumab, HNK-20, palivizumab,RespiGam, and anti-staphylococcus antibodies such as Aurexis, Aurograb,BSYX-A110, and SE-Mab.

Alternatively, the antibodies of the present invention may beco-administered or with one or more other molecules that compete forbinding to one or more Fc receptors. For example, co-administeringinhibitors of the inhibitory receptor FcγRIIb may result in increasedeffector function. Similarly, co-administering inhibitors of theactivating receptors such as FcγRIIIa may minimize unwanted effectorfunction. Fc receptor inhibitors include, but are not limited to, Fcmolecules that are engineered to act as competitive inhibitors forbinding to FcγRIIb FcγRIIIa, or other Fc receptors, as well as otherimmunoglobulins and specifically the treatment called IVIg (intravenousimmunoglobulin). In one embodiment, the inhibitor is administered andallowed to act before the antibody is administered. An alternative wayof achieving the effect of sequential dosing would be to provide animmediate release dosage form of the Fc receptor inhibitor and then asustained release formulation of the antibody of the invention. Theimmediate release and controlled release formulations could beadministered separately or be combined into one unit dosage form.Administration of an FcγRIIb inhibitor may also be used to limitunwanted immune responses, for example anti-Factor VIII antibodyresponse following Factor VIII administration to hemophiliacs.

In one embodiment, the antibodies of the present invention areadministered with a chemotherapeutic agent. By “chemotherapeutic agent”as used herein is meant a chemical compound useful in the treatment ofcancer. Examples of chemotherapeutic agents include but are not limitedto alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan; androgenssuch as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimax,zinostatin, zorubicin; anti estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY 117018, onapristone, and toremifene(Fareston); anti-metabolites such as methotrexate and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; folic acidreplenisher such as frolinic acid; nitrogen mustards such aschlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; platinum analogs such ascisplatin and carboplatin; vinblastine; platinum; proteins such asarginine deiminase and asparaginase; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; taxanes,e.g. paclitaxel (TAXOL®; Bristol-Myers Squibb Oncology, Princeton, N.J.)and docetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France);topoisomerase inhibitor RFS 2000; thymidylate synthase inhibitor (suchas Tomadex); additional chemotherapeutics including aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabacil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;difluoromethylornithine (DMFO); elformithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitrobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; retinoic acid; esperamicins; capecitabine.Pharmaceutically acceptable salts, acids or derivatives of any of theabove may also be used.

A chemotherapeutic or other cytotoxic agent may be administered as aprodrug. By “prodrug” as used herein is meant a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic totumor cells compared to the parent drug and is capable of beingenzymatically activated or converted into the more active parent form.See, for example Wilman, 1986, Biochemical Society Transactions, 615thMeeting Belfast, 14:375-382; Stella et al., “Prodrugs: A ChemicalApproach to Targeted Drug Delivery,” Directed Drug Delivery; andBorchardt et al., (ed.): 247-267, Humana Press, 1985, all incorporatedentirely by reference. The prodrugs that may find use with the presentinvention include but are not limited to phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, beta-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use with the antibodies of thepresent invention include but are not limited to any of theaforementioned chemotherapeutic agents.

A variety of other therapeutic agents may find use for administrationwith the antibodies of the present invention. In one embodiment, theantibody is administered with an anti-angiogenic agent. By“anti-angiogenic agent” as used herein is meant a compound that blocks,or interferes to some degree, the development of blood vessels. Theanti-angiogenic factor may, for instance, be a small molecule or aprotein, for example an antibody, Fc fusion, or cytokine, that binds toa growth factor or growth factor receptor involved in promotingangiogenesis. The preferred anti-angiogenic factor herein is an antibodythat binds to Vascular Endothelial Growth Factor (VEGF). Other agentsthat inhibit signaling through VEGF may also be used, for exampleRNA-based therapeutics that reduce levels of VEGF or VEGF-R expression,VEGF-toxin fusions, Regeneron's VEGF-trap, and antibodies that bindVEGF-R. In an alternate embodiment, the antibody is administered with atherapeutic agent that induces or enhances adaptive immune response, forexample an antibody that targets CTLA-4. Additional anti-angiogenesisagents include, but are not limited to, angiostatin (plasminogenfragment), antithrombin III, angiozyme, ABT-627, Bay 12-9566, benefin,bevacizumab, bisphosphonates, BMS-275291, cartilage-derived inhibitor(CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, combretastatinA-4, endostatin (collagen XVIII fragment), farnesyl transferaseinhibitors, fibronectin fragment, gro-beta, halofuginone, heparinases,heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin(hCG), IM-862, interferon alpha, interferon beta, interferon gamma,interferon inducible protein 10 (IP-10), interleukin-12, kringle 5(plasminogen fragment), marimastat, metalloproteinase inhibitors (eg.TIMPs), 2-methodyestradiol, MMI 270 (CGS 27023A), plasminogen activatorinhibitor (PAI), platelet factor-4 (PF4), prinomastat, prolactin 16 kDafragment, proliferin-related protein (PRP), PTK 787/ZK 222594,retinoids, solimastat, squalamine, SS3304, SU5416, SU6668, SU11248,tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1(TSP-1), TNP-470, transforming growth factor beta (TGF-β),vasculostatin, vasostatin (calreticulin fragment), ZS6216 and ZD6474.

In a preferred embodiment, the antibody is administered with tyrosinekinase inhibitor. By “tyrosine kinase inhibitor” as used herein is meanta molecule that inhibits to some extent tyrosine kinase activity of atyrosine kinase. Examples of such inhibitors include but are not limitedto quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazoline;pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines; such as CGP59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo(2,3-d) pyrimidines; curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lambert); antisensemolecules (e.g. those that bind to ErbB-encoding nucleic acid);quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No.5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering A G);pan-ErbB inhibitors such as C1-1033 (Pfizer); Affinitac (ISIS 3521;Isis/Lilly); Imatinib meslyate (STI571, Gleevec®; Novartis); PKI 166(Novartis); GW2016 (Glaxo SmithKline); C1-1033 (Pfizer); EKB-569(Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-IC11 (Imclone); or as described in any ofthe following patent publications: U.S. Pat. No. 5,804,396; PCT WO99/09016 (American Cyanimid); PCT WO 98/43960 (American Cyanamid); PCTWO 97/38983 (Warner-Lamber); PCT WO 99/06378 (Warner-Lambert); PCT WO99/06396 (Warner-Lamber); PCT WO 96/30347 (Pfizer, Inc); PCT WO 96/33978(AstraZeneca); PCT WO96/3397 (AstraZeneca); PCT WO 96/33980(AstraZeneca), gefitmib (IRESSA™, ZD1839, AstraZeneca), and OSI-774(Tarceva™, OSI Pharmaceuticals/Genetech), all patent publicationsincorporated entirely by reference.

In another embodiment, the antibody is administered with one or moreimmunomodulatory agents. Such agents may increase or decrease productionof one or more cytokines, up- or down regulate self-antigenpresentation, mask MHC antigens, or promote the proliferation,differentiation, migration, or activation state of one or more types ofimmune cells. Immunomodulatory agents include but not limited to:non-steroidal anti-inflammatory drugs (NSAIDs) such as asprin,ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin,ketoralac, oxaprozin, nabumentone, sulindac, tolmentin, rofecoxib,naproxen, ketoprofen, and nabumetone; steroids (eg. glucocorticoids,dexamethasone, cortisone, hydroxycortisone, methylprednisolone,prednisone, prednisolone, trimcinolone, azulfidincicosanoids such asprostaglandins, thromboxanes, and leukotrienes; as well as topicalsteroids such as anthralin, calcipotriene, clobetasol, and tazarotene);cytokines such as TGFb, IFNa, IFNb, IFNg, IL-2, IL-4, IL-10; cytokine,chemokine, or receptor antagonists including antibodies, solublereceptors, and receptor-Fc fusions against BAFF, B7, CCR2, CCR5, CD2,CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28,CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, CD147, CD152,complement factors (C5, D) CTLA4, eotaxin, Fas, ICAM, ICOS, IFNα, IFNβ,IFNγ, IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA-1, LFA-3,MHC, selectins TGFβ, TNFα, TNFβ, TNF-R1, T-cell receptor, includingEnbrel® (etanercept), Humira® (adalimumab), and Remicade® (infliximab);heterologous anti-lymphocyte globulin; other immunomodulatory moleculessuch as 2-amino-6-aryl-5 substituted pyrimidines, anti-idiotypicantibodies for MHC binding peptides and MHC fragments, azathioprine,brequinar, bromocryptine, cyclophosphamide, cyclosprine A,D-penicillamine, deoxyspergualin, FK506, glutaraldehyde, gold,hydroxchloroquine, leflunomide, malononitriloamides (eg. leflunomide),methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin,and sulfasasazine.

In an alternate embodiment, antibody of the present invention areadministered with a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intracellular mediators. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormone such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, beta, and -gamma; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, a tumor necrosisfactor such as TNF-alpha or TNF-beta; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell culture,and biologically active equivalents of the native sequence cytokines.

In a preferred embodiment, cytokines or other agents that stimulatecells of the immune system are co-administered with the antibody of thepresent invention. Such a mode of treatment may enhance desired effectorfunction. For example, agents that stimulate NK cells, including but notlimited to IL-2 may be co-administered. In another embodiment, agentsthat stimulate macrophages, including but not limited to C5a, formylpeptides such as N-formyl-methionyl-leucyl-phenylalanine(Beigier-Bompadre et al. (2003) Scand. J. Immunol. 57:221-8,incorporated entirely by reference), may be co-administered. Also,agents that stimulate neutrophils, including but not limited to G-CSF,GM-CSF, and the like may be administered. Furthermore, agents thatpromote migration of such immunostimulatory cytokines may be used. Alsoadditional agents including but not limited to interferon gamma, IL-3and IL-7 may promote one or more effector functions.

In an alternate embodiment, cytokines or other agents that inhibiteffector cell function are co-administered with the antibody of thepresent invention. Such a mode of treatment may limit unwanted effectorfunction.

In an additional embodiment, the antibody is administered with one ormore antibodies, including but not limited to: aminoglycosideantibiotics (eg. apramycin, arbekacin, bambermycins, butirosin,dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,ribostamycin, sisomycin, spectrinomycin), aminocyclitols (eg.spretinomycin), amphenicol antibiotics (eg. azidamfenicol,chloramphenicol, florfrnicol, and thiamphemicol), ansamycin antibiotics(eg. rifamide and rifampin), carbapenems (eg. imipenem, meropenem,panipenem); cephalosporins (eg. cefaclor, cefadroxil, cefamandole,cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide,cefpirome, cefprozil, cefuroxine, cefixime, cephalexin, cephradine),cephamycins (cefbuperazone, cefoxitin, cefminox, cefmetazole, andcefotetan); lincosamides (eg. clindamycin, lincomycin); macrolide (eg.azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin,tobramycin), monobactams (eg. aztreonam, carumonam, and tigemonam);mupirocin; oxacephems (eg. flomoxef, latamoxef, and moxalactam),penicillins (eg. amdinocillin, amdinocillin pivoxil, amoxicillin,bacampicillin, bexzylpenicillinic acid, benzylpenicillin sodium,epicillin, fenbenicillin, floxacillin, penamecillin, penethamatehydriodide, penicillin o-benethamine, penicillin O, penicillin V,penicillin V benzoate, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium); polypeptides (eg. bacitracin, colistin,polymixin B, teicoplanin, vancomycin); quinolones (amifloxacin,cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, feroxacin, flumequine,gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, perfloxacin,pipemidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin,tosulfoxacin, trovafloxacin); rifampin; streptogramins (eg.quinupristin, dalfopristin); sulfonamides (sulfanilamide,sulfamethoxazole); tetracyclenes (chlortetracycline, demeclocyclinehydrochloride, demethylchlortetracycline, doxycycline, duramycin,minocycline, neomycin, oxytetracycline, streptomycin, tetracycline,vancomycin).

Anti-fungal agents such as amphotericin B, ciclopirox, clotrimazole,econazole, fluconazole, flucytosine, itraconazole, ketoconazole,niconazole, nystalin, terbinafine, terconazole, and tioconazole may alsobe used.

Antiviral agents including protease inhibitors, reverse transcriptaseinhibitors, and others, including type I interferons, viral fusioninhibitors, and neuramidase inhibitors, may also be used. Examples ofantiviral agents include, but are not limited to, acyclovir, adefovir,amantadine, amprenavir, clevadine, enfuvirtide, entecavir, foscamet,gangcyclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin,rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, andzidovudine, may be used.

The antibodies of the present invention may be combined with othertherapeutic regiments. For example, in one embodiment, the patient to betreated with an antibody of the present invention may also receiveradiation therapy. Radiation therapy can be administered according toprotocols commonly employed in the art and known to the skilled artisan.Such therapy includes but is not limited to cesium, iridium, iodine, orcobalt radiation. The radiation therapy may be whole body irradiation,or may be directed locally to a specific site or tissue in or on thebody, such as the lung, bladder, or prostate. Typically, radiationtherapy is administered in pulses over a period of time from about 1 to2 weeks. The radiation therapy may, however, be administered over longerperiods of time. For instance, radiation therapy may be administered topatients having head and neck cancer for about 6 to about 7 weeks.Optionally, the radiation therapy may be administered as a single doseor as multiple, sequential doses. The skilled medical practitioner candetermine empirically the appropriate dose or doses of radiation therapyuseful herein. In accordance with another embodiment of the invention,the antibody of the present invention and one or more other anti-cancertherapies are employed to treat cancer cells ex vivo. It is contemplatedthat such ex vivo treatment may be useful in bone marrow transplantationand particularly, autologous bone marrow transplantation. For instance,treatment of cells or tissue(s) containing cancer cells with antibodyand one or more other anti-cancer therapies, such as described above,can be employed to deplete or substantially deplete the cancer cellsprior to transplantation in a recipient patient.

It is of course contemplated that the antibodies of the invention mayemploy in combination with still other therapeutic techniques such assurgery of phototherapy.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation.

For reference to immunoglobulin variable regions, positions are numberedaccording to the Kabat numbering scheme. For reference to immunoglobulinconstant regions, positions are numbered according to the EU index as inKabat (Kabat et al., 1991, Sequence of Proteins of ImmunologicalInterest, 5th Ed., United States Public Health Service, NationalInstitute of Health, Bethesda).

Example 1. Anti-CD19 Antibodies with Amino Acid Modifications thatEnhance Effector Function

The anti-CD19 antibodies of the invention are intended as clinicalcandidates for anti-cancer therapeutics. To investigate the possibilityof improving the effector function of an antibody that targets CD19,variant versions of anti-CD19 antibodies were engineered.

FIG. 6 provides some heavy and light chain variable region sequences ofthe anti-CD19 antibodies 4G7 (Meeker, T. C. et al. 1984, Hybridoma, 3:305-320) and HD37 (Pezzuto, A. et al. 1987, J. Immunol. 138: 2793-2799)used in the present study. The mouse, patient chimeric heavy and lightchains are labeled H0 4G7, H0 HD37, L0 4G7, and L0 HD37 respectively.Variants of the present invention could also be made in the context ofthe anti-CD19 antibody B43 (Uckun, F. M. et al., 1998, Blood, 71: 13-29)which has similar properties to HD37 and shares identical CDRs and anoverall 97% sequence identity relative to the HD37 H0 and L0 sequencesshown in FIG. 6. The genes for marine WT 4G7 and HD37 VH and VL,designated H0 and L0 respectively, were constructed using gene synthesistechniques and subcloned into the mammalian expression vectorpcDNA3.1Zeo (Invitrogen) comprising the full length light kappa (Cκ) andheavy chain IgG1 constant regions. Variant S239D/I332E (effectorfunction enhanced anti-CD19) was constructed in the Fc region of ahybrid IgG1/IgG2 (referred to as “Hybrid”, FIG. 2) antibody in thepcDNA3.1Zeo vector using QuikChange mutagenesis techniques (Stratagene).All sequences were sequenced to confirm the fidelity of the sequence.Plasmids containing heavy chain gene (VH-CH1-CH2-CH3) (wild type orvariants) were co-transfected with plasmid containing light chain gene(VL-CLκ) into 293T cells. Media were harvested 5 days aftertransfection, and antibodies were purified from the supernatant usingprotein A affinity chromatography (Pierce, Catalog #20334).

The relative binding affinities of 4G7 Hybrid S239D/I332E and 4G7 IgG1antibody were calculated by determining binding parameters on Biacore™using a panel of Fc receptors (FIG. 7). Briefly, protein A/G was coupledto a flow cell of a CM5 chip. IgG was first diluted to 25 nM andimmobilized to protein A/G channel to ˜1000 RUs. FcγR-His was seriallydiluted and injected at 30 mL/min for 2 min followed by dissociation for3 min. To determine KD the resulting sensorgrams are “group-fitted”using the 1:1 interaction model available in BIAevaluation software.Values of K_(D) that were higher than 5×10⁻⁶ M are labeled as ND (notdetermined) in FIG. 7. The data indicate that WT IgG1 antibody bindsV158 FcγRIIIa with an affinity of approximately 240 nM, consistent withthe literature (Okazaki et al, 2004, J Mol Bio 336:1239-49; Lazar et al,Proc Natl Acad Sci USA 103(11):4005-4010). The Fc variant version bindswith an affinity to V158 FcγRIIIa of about 4.7 nM, indicating anaffinity enhancement of about 50-fold relative to WT. Binding of variantanti-CD19 to F158 FcγRIIIa is about 16.7 nM.

To assess the capacity of the antibody variants to mediate effectorfunction against CD19 expressing cells, effector function enhancedanti-CD19 was tested in a cell-based ADCC assay. Human peripheral bloodmonocytes (PBMCs) were isolated from leukopaks and used as effectorcells, and CD19 positive cancer cells were used as target cells. Targetcells were seeded at 10,000 (Raji and MEC-1) and 20,000 (SUP-B15)cells/well in 96-well plates and treated with designated antibodies intriplicates. PBMCs isolated using a Ficoll gradient were added in excessto target cells and co-cultured for 4 hrs before processing for LDHactivity using the Cytotoxicity Detection Kit according to themanufacturer's instructions. FIG. 8a shows the results of the ADCC assaycomparing 4G7 IgG1 and 4G7 Hybrid S239D/I332E antibodies, and HD37 IgG1and HD37 Hybrid S239D/I332E on the cell line Daudi (BL). FIG. 8b showsthe results of the ADCC assay comparing 4G7 IgG1 and 4G7 HybridS239D/I332E antibodies, and anti-CD20 rituximab on the cell linesSUP-B15 (ALL) and Raji (Burkitt's Lymphoma). The graphs show that theantibodies differ not only in their EC50, reflecting their relativepotency, but also in the maximal level of ADCC attainable by theantibodies at saturating concentrations, reflecting their relativeefficacy. Considerable enhancements in potency and efficacy are observedfor the Fc variant antibodies as compared to the antibody with WT Fcregion. The chimeric IgG1 antibody has very little efficacy or potency.

EC50 of a dose response curve such as that in FIG. 8 represents theconcentration of a compound where 50% of its maximal effect is observed.In a clinical setting, potency reflects the concentration of antibodyneeded to carry out its therapeutic effect. Thus the data in FIG. 8 showthat the Fc optimized anti-CD19 antibodies act in vivo at aconcentration or dose lower than that of a WT anti-CD19 or anti-CD20antibody. In FIG. 8b , whereas WT IgG1 anti-CD19 at saturatingconcentration mediates approximately 10% maximal ADCC, Fc variantanti-CD19 lyses approximately 60% of the target cells. In a clinicalsetting, efficacy reflects the maximal therapeutic benefit from theadministered drug.

Example 2. Binding of an Effector Function Enhanced Anti-CD19 Antibodyto a B-cell Derived Tumor Cell Line

The relative binding of 4G7 Hybrid S239D/I332E to the Raji cell line wasmeasured. Affinities of enhanced effector function anti-CD19 variantswere determined by using the DELFIA® system (PerkinElmer Life Sciences)which is based on Time-Resolved Fluorometry (TRF). Anti-CD19 (H0L0) islabeled with Europium using the Eu-Labeling kit available fromPerkinElmer Biosciences. Unlabeled wild-type (WT) or variants (cold) areserially diluted (typically starting from 1 nM) in ½ log steps and mixedwith a fixed concentration of labeled (or hot) anti-CD19. The mix of“hot” and “cold” antibodies are then added to 100,000 Raji Cells (thathave a high density of surface expressed CD19 antigen) and incubated onice for 30 min. The assay is essentially applied as a competition assayfor screening anti-CD19 antibodies of different affinities. In theabsence of competing affinity variants, Eu-anti-CD19 and surface CD19interact and produce a signal at 613 nm when the Europium is excited at340 nm. Addition of wild type or variant competes with Eu-anti-CD19-CD19interaction, reducing fluorescence quantitatively to enabledetermination of relative binding affinities. FIG. 9 shows results of acell-surface binding assay of enhanced effector function anti-CD19 toRaji cells. As can be seen, the calculated EC50 value is 1.2 nM.

Example 3. ADCC of an Anti-CD19 Antibody with Enhanced CytotoxicityAgainst Multiple Lymphoma Cell Lines

In order to evaluate cytotoxic properties of effector function enhancedanti-CD19, ADCC assays were performed on a panel of 14 cell linesrepresenting various lymphomas and leukemias (FIG. 10a ). Cell linestested were the Follicular Lymphoma (FL) cell lines DoHH-2 and SCI;Mantle Cell Lymphoma (MCL) cell line Jeko-1: Burkitt's Lymphoma (BL)cell lines Daudi and Raji; Chronic Lymphocytic Leukemia (CLL) cell linesMEC1 and WaC3CD5; Hairy Cell Leukemia (HCL) cell line Bonna-12; ChronicMyelogenous Leukemia (CML) cell line BV-173; and Acute LymphoblasticLeukemia (ALL) cell lines VAL, SUP-B15, NALM-6, RS4;11, and 697. Humanperipheral blood monocytes (PBMCs) were isolated from leukopaks and usedas effector cells, and CD19 positive cancer cells were used as targetcells. Target cells were seeded in 96-well plates and treated withdesignated antibodies in triplicate. PBMCs isolated using a Ficollgradient were added in excess to target cells and co-cultured for 4 hrsbefore processing for LDH activity using the Cytotoxicity Detection Kitaccording to the manufacturer's instructions. Both parameters, potency(EC50) and efficacy (% ADCC) were normalized to that of rituximab(anti-CD20). This screen has demonstrated the cytotoxic superiority invitro of effector function enhanced anti-CD19 over a broad range of celllines, especially representing the lympho-proliferative disease thatoriginates in early stages of B cell development. FIG. 10b lists celllines used and their corresponding cancer type.

Example 4. Anti-CD19 Antibodies with Reduced Potential forImmunogenicity

Due to the wide use of hybridoma technology, a substantial number ofantibodies are derived from nonhuman sources. However, nonhuman proteinsare often immunogenic when administered to humans, thereby greatlyreducing their therapeutic utility. Immunogenicity is the result of acomplex series of responses to a substance that is perceived as foreign,and may include production of neutralizing and non-neutralizingantibodies, formation of immune complexes, complement activation, mastcell activation, inflammation, hypersensitivity responses, andanaphylaxis. Several factors can contribute to protein immunogenicity,including but not limited to protein sequence, route and frequency ofadministration, and patient population. Immunogenicity may limit theefficacy and safety of a protein therapeutic in multiple ways. Efficacycan be reduced directly by the formation of neutralizing antibodies.Efficacy may also be reduced indirectly, as binding to eitherneutralizing or non-neutralizing antibodies typically leads to rapidclearance from serum. Severe side effects and even death may occur whenan immune reaction is raised. Thus in a preferred embodiment, proteinengineering is used to reduce the immunogenicity of the CD19 targetingproteins of the present invention.

In order to reduce the potential for immunogenicity of the anti-CD19proteins of the present invention, the immunogenicity of the anti-CD19antibodies 4G7 and HD37 were reduced using a method described in U.S.Ser. No. 60/619,483, filed Oct. 14, 2004 and U.S. Ser. No. 11/004,590,entitled “Methods of Generating Variant Proteins with Increased HostString Content and Compositions Thereof”, filed on Dec. 6, 2004. Themethods reduce the potential for immunogenicity by increasing the humanstring content of the antibody through mutations. The heavy and lightchains with reduced potential for immunogenicity are named H1, H2, H3,H4, etc. and L1, L2, L3, etc. and are shown in FIGS. 11 thru 14. Theheavy and light chains of the original antibodies, 4G7 and HD37, arereferred to as H0 and L0, respectively. Combinations of the differentheavy and light chains were expressed and the resulting antibodies, withnames such as H3L3, H3/L3, or H3_L3, were purified and examined.Anti-CD19 antibodies were expressed by transient transfection of vectorsencoding the heavy and light chains into 293T cells grown in 10% ultralow IgG fetal bovine serum with 1 mM sodium pyruvate and 1×non-essential amino acids (Gibco®, Invitrogen Hayward Calif.). Five daysafter transfection, the culture media was removed and passed through aprotein A column (Pierce Biotechnology Inc, Rockford Md.). The heavychains may be made with any type of constant domain including, inhumans, IgG1, IgG2 and hybrids comprising IgG1 and IgG2 as well as mouseconstant domains such as IgG1 and IgG2a, which may be referred to asmIgG1 and mIgG2a. The sequences of human heavy chains may be found inFIG. 2. The relative binding of anti-CD19 variants with reducedimmunogenicity to the Raji cell line was measured. Affinities of reducedimmunogenicity anti-CD19 CD19 variants were determined by using theDELFIA® system (PerkinElmer Life Sciences) which is based onTime-Resolved Fluorometry (TRF). Anti-CD19 is labeled with Europiumusing the Eu-Labeling kit available from PerkinElmer Biosciences.Unlabeled wild-type (WT) or variants (cold) are serially-diluted(typically starting from 1 uM) in ½ log steps and mixed with a fixedconcentration of labeled (or hot) anti-CD19. The mix of “hot” and “cold”antibodies are then added to 100,000 Raji Cells (that have a highdensity of surface expressed CD-19 antigen) and incubated on ice for 30min. The assay is essentially applied as a competition assay forscreening anti-CD19 antibodies of different affinities. In the absenceof competing affinity variants, Eu-anti-CD19 and surface CD19 interactand produce a signal at 613 nm when the Europium is excited at 340 nm.Addition of wild type or variant competes with Eu-anti-CD19-CD19interaction, reducing fluorescence quantitatively to enabledetermination of relative binding affinities. FIG. 15a shows results ofa cell-surface binding assay of reduced immunogenicity 4G7 variants toRaji cells. Based on binding affinity and stability, the variable region4G7 H1L1 was chosen for further development. FIG. 15b shows results ofan ADCC assay on reduced immunogenicity templates HD37_H2L1 HybridS239D/I332E and 4G7_H1L3 Hybrid S239D/I332E on the cell line MEC-1(CLL). This ADCC assay was performed as in the previous assays. Bothantibodies are active on this cell line and therefore may be potentialtreatments for CLL.

Example 5. Affinity and Stability Enhancement of Effector FunctionEnhanced Anti-CD19

Affinity maturation of 4G7 mAb H1L1 was carried out in order to furtherincrease CD19 binding affinity as well as ADCC potency. The affinitymaturation was performed in three stages using a computational/proteinengineering approach. First, operating under the hypothesis that thespecificity determining residues (SDRs) (Padlan, E. A. et al. 1995,FASEB J. 9: 133-139) in the CDRs of an antibody have already beenoptimized by B-cells in the process of in vivo somatic hypermutation, alibrary of 94 variants was designed to determine those residues in theCDRs that were critical for antigen binding, and thus should not bechanged during the engineering process. This library consisted of one ortwo “probing” mutations made at positions in the CDRs with sites chosenusing structural modeling as well as the likelihood that a position isoften an SDR, which was compiled from analysis of availableantigen-antibody complex structures in the Protein Data Bank (PDB)(MacCallum, R. M. et al. 1996. JMB 262: 732-745; Almagro, J. C. 2004. J.Mol. Recognit. 17:132-143).

Variant mutations were introduced using the QuikChange mutagenesis kitin the Fab format of the H1L1 template and contained a 6×-His tag.Variant Fabs were expressed in 293T cells using 24-well plates and wereanalyzed by Alpha Screen or flow cytometry using Raji or RS4;11 cells,and with the concentration of each variant determined using aHis-binding chip by Biacore™. Out of 50 positions, 17 positions wereidentified that were critical to antigen binding, enabling us to reducethe library size in the next round of affinity maturation and giving usvaluable structural information as to which positions lie close to theantigen interface and would make good targets for finding increasedaffinity variants. The 17 SDRs identified in our analysis are inexcellent agreement with the average number of SDRs present inantibodies whose antigen-antibody complexes have been solved (Almagro,J. C. 2004. J. Mol. Recognit. 17:132-143). In addition to the valuablestructural information gained from this library, some variants wereobtained that had an increased affinity.

The remaining 33 CDR positions were ranked in order of importance basedon analysis of the first library results and by mapping the SDRs onto astructural model of the H1L1 template. Through this analysis it wasdetermined that nearly the entire antigen-antibody binding interfacecould be explored with a total frequency of 12.2 amino acids perposition (˜9.3 new variants per position) with a second round librarysize of 279 variants. Library Design Automation (LDA™) (U.S. Ser. No.11/367,184, filed Mar. 3, 2006) was used to design an optimized libraryof variants that was tuned for both fitness and coverage based on thenumber of variants desired. The final second round library when adjustedfor high-throughput format contained 265 variants at 30 positions. Thislibrary yielded several variants displaying increased binding affinity.Anti-CD19 Fab variants were screened by flow cytometry to determine theaffinity. The cell line RS4;11, known to express CD19, were suspended inPBS and plated at 200,000 cells/well in a 96-well round bottom plate. Aserial dilution of CD19 antibodies were added to the RS4;11 cells at anunknown concentration. The cells were incubated on ice for 30 minutesand then washed 4 times in PBS. An anti-Fab PE-labeled F(ab′)₂ wasdiluted 1/50 in PBS, which was then used to resuspend the anti-CD19 Fabcoated RS4;11. Cells were incubated for 30 minutes and washed two times.The cells were then fixed and the binding assay was evaluated on a FACSCanto II flow cytometer. The MFI was used to measure the tightness ofbinding. From both libraries one and two, a total of 30 increasedaffinity single variants were obtained at 11 positions.

Analysis of the binding data from the first two libraries as well asfurther structural analysis enabled us to design a third and finallibrary containing combinations of 2-8 single variants. This libraryconsisted of 149 variants at 8 positions. From these, 20 variants showeda significant increase in affinity and were selected for conversion tofull length format for simultaneous measurement of binding affinity andADCC. To assess solution properties, stability assays on these variantswere performed. The final set of mutations included in the final 20 wereheavy chain variants T57P, K58E, S100cT, R100dS, and light chainvariants L27cQ, S27cV, A55N, F96I, and F96N. Accelerated stabilitystudies revealed that at least one of the affinity enhancing mutationscreated instability in the protein and caused these variants to lose allpotency after only 8 hrs at 37° C. Taking the binding and stability datainto account, a final affinity matured candidate mAb was able to beselected which displayed an ˜10-fold increase in binding affinity onRS4;11 cells relative to the H1L1 mAb (FIG. 16). Variants designed toincrease the long-term stability of the anti-CD19 molecule were alsodesigned and screened. FIG. 17 shows binding data for variants incubatedfor 5 days at 37° C., pH 9.0 in 200 mM Tris-HCl, demonstrating theimprovement in stability obtained from an anti-CD19 variant.

All single substitutions made for enhanced stability and/or affinity areshown in FIG. 27. FIG. 28 lists all anti-CD19 variable region variantsconstructed to optimize affinity and stability. FIG. 29 lists preferredvariants and relative increase in binding affinity versus the parentH1L1 mAb. Sequences for the preferred affinity and/or stability enhancedheavy chain variants are shown in FIG. 18. Sequences for the preferredaffinity and/or stability enhanced light chain variants are shown inFIG. 19. Amino acid sequences of full length hybrid S239D/I332E variantscontaining the affinity and stability improved variable regions areprovided as SEQ ID NOs: 86-110. Affinity and stability improved CDR'sare provided as SEQ ID NOs: 111-131.

Example 6. Anti-Proliferative Properties of 4G7 Hybrid S239D/I332 onRaji Cells

To observer an anti-proliferative effect in vitro, many antibodiesrequire cross-linking, usually accomplished by a secondary antibody. Ithas been proposed that corresponding in vivo effects for theseantibodies may be dependent on cross-linking mediated by Fc receptorsexpressed on the surface of effector cells. In this experiment Rajicells were grown for 3 days in the presence of 100 nm/mL 4G7 HybridS239D/I332E, 4G7 IgG1, or anti-CD20 (rituximab) or control antibodies(non-CD19 binding variable region with Hybrid S239D/I332E variants Fc)at varying concentrations with 10× molar excess of cross-linkingantibody. Cell growth was measured using an ATP-dependent luminescenceassay. Results for the anti-proliferation assay are shown in FIG. 20.Both 4G7 Hybrid S239D/I332E and 4G7 IgG1 show strongeranti-proliferation effects than rituximab.

Example 7. Anti-Proliferative Properties of 4G7 Stability and AffinityImproved Hybrid S239D/I332E on SU-DHL-6 Cells

In this experiment SU-DHL-6 cells were either grown for 3 days in thepresence of humanized 4G7 stability and affinity improved HybridS239D/I332E and control antibodies at varying concentrations with 10×molar excess of cross-linking antibody and 600 cells/well or were grownin the presence of a fixed concentration of antibody at 3000 cells/welland viability at specific time points measured for a total of 72 hours.Results for the anti-proliferation assay are shown in FIG. 21, 4G7stability and affinity improved Hybrid S239D/I332E shows strongeranti-proliferative effects than rituximab. 4G7 stability and affinityimproved Hybrid S239D/I332E also shows anti-proliferative effects evenin the absence of cross-linking antibody.

Example 8. Phagocytosis of Raji and RS4;11 Cells with 4G7 Stability andAffinity Improved Hybrid S239D/I332E

Unlike NK cells which only express FcγRIIIa and sometimes FcγRIIc,monocytes and monocyte-derived effector cells express the range ofFcγRs, including FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa. Thus theactivation and function of monocyte-derived effector cells, includingfor example macrophages, may be dependent on engagement of antibodyimmune complexes with receptors other than only FcγRIIIa. Indeed asdescribed in PCT/US2006/038842, Desjarlais, J. R. et al., filed Oct. 3,2006, phagocytosis by macrophages is mediated in part by engagement ofantibody with FcγRIIa.

To assess the ability of 4G7 stability and affinity improved HybridS239D/I332E to mediate phagocytosis a flow cytometry based phagocytosisassay was performed. Purified CD14⁺ monocytes were cultured inmacrophage colony stimulating factor (50 ng/ml) for 5 days in ahumidified incubator to differentiate macrophages, RS4;11 or Raji cellswere used as targets. The target cells were labeled with PKH67 (Sigma)according to the manufacture's instructions. Cells were added to a 96well plate after which a serial dilution of WT and Fc modified anti-CD19antibodies were added. Monocyte-derived macrophages were then added tothe wells at an effector to target ratio of 4:1. These assays wereperformed in the presence of human serum. The co-culture of cells werebriefly spun down and then incubated in a humidified incubator for 4hours. The cells were harvested, and macrophages were stained with asecond fluorescent color to distinguish them from the target. The cellswere fixed in 1% PFA and phagocytosis was evaluated on a FACS Canto IIflow cytometer. The read out of phagocytosis was determined by thenumber of double positive cells divided by the total number of tumorcells. Results of the phagocytosis assay are shown in FIG. 22. 4G7stability and affinity improved Hybrid S239D/I332E shows an increasedlevel of phagocytosis on both cell lines compared to the IgG1 anti-CD19antibody.

Macrophages are phagocytes that acts as scavengers to engulf dead cells,foreign substances, and other debris. Importantly, macrophages areprofession antigen presenting cells (APCs), taking up pathogens andforeign structures in peripheral tissues, then migrating to secondarylymphoid organs to initiate adaptive immune responses by activatingnaive T-cells. Thus the results of the previous experiment suggest thatmodification of anti-CD19 antibodies may enable mechanisms of actionthat include both innate cytotoxic effector functions, as well aseffector functions that can potentially lead to long-term adaptiveimmune response.

Example 9. ADCC of 4G7 Stability and Affinity Improved HybridS239D/I332E Against Multiple Lymphoma Cell Lines Using Purified NaturalKiller (NK) Cells

In order to evaluate cytotoxic properties of 4G7 stability and affinityimproved Hybrid S239D/I332E, ADCC assays were performed with purified NKcells on a panel of 6 cell lines representing various lymphomas andleukemias (FIG. 23). ADCC with purified NK cells is done in 96-wellmicrotiter plates. The NK cells were purified from human PBMC using thekit from Miltenyi Biotec (Cat #130-091-152) and incubated in 10%FBS/RPMI1640 overnight with 10 ng/ml IL-2. The following day, 10,000(WaC3CD5, Namalwa, Bonna-12, Ramos) or 20,000 (RS4;11, BV-173) cancertarget cells are opsonized with varying concentrations of antibody and50 k NK cells are used for each antibody concentration in triplicates.The target cells are washed three times while NK cells are washed twicewith RPMI1640 and both resuspended in 1FBS/RPMI1640 and added to theantibody solutions. After 4 hours of incubation at 37° C. in ahumidified incubator with 5% CO₂, the assay was quantified using LDHdependent CytoTox-One fluorescence dependent detection system fromPromega (#PAG7891). Total LDH signal is determined from the Triton-X100lysed target cells (total Target LDH) and used to normalize against thespontaneous LDH background (Spontaneous Background) adjustedexperimental values. Thus % ADCC=((Experimental Value−SpontaneousBackground)/(Total Target LDH−Target LDH))*100. Spontaneous backgroundis the value obtained from the Target and NK cells co-incubated in theabsence of antibody. Target LDH is the value from the target cancercells alone spontaneously releasing LDH during the incubation. FIG. 23shows results of the ADCC assay for 6 cell lines using 4G7 stability andaffinity improved Hybrid S239D/I332E, 4G7 IgG1 (with affinity/stabilityoptimized variable region), rituximab (anti-CD20), and an isotypecontrol antibody. For all cell lines tested, 4G7 stability and affinityimproved Hybrid S239D/I332E performs better in both potency and efficacywhen compared to 4G7 IgG1 and rituximab.

Example 10. 4G7 Stability and Affinity Improved Hybrid S239D/I332EBinding to CD19 Transfected 293T Cells

A human CD19 clone was ordered from Origene (catalog No. SC127938) andtransfected into 293T cells. Cells were suspended in PBS and plated at100 000 cells/well. A serial dilution of 4G7 stability and affinityimproved Hybrid S239E/I332E was added to the cells and then the cellswere incubated on ice for 30 minutes and then washed 4 times in PBS. Ananti-Fab PE-labeled F(ab′)₂ was diluted 1/50 in PBS, which was then usedto resuspend the 4G7 stability and affinity improved Hybrid S239D/I332Eanti-CD19 coated 293T cells. Cells were incubated for 30 minutes andwashed two times. The cells were then fixed and the binding wasevaluated on a FACS Canto II flow cytometer. FIG. 24 displays resultsfor this assay. The results show that 4G7 stability and affinityimproved Hybrid S239D/I332E binds to 293T cells transfected with CD19and does not bind to the control cells (normal 293T cells).

Example 11. 4G7 Stability and Affinity Improved Hybrid S239D/I332E isCross-section with CD19 from Cynomolgus and Rhesus Monkeys

Pre-clinical testing of drugs in monkeys is typically an important stepin drug discovery in order to assess potential toxicity. Blood samplesfrom five cynomolgus (Macaca fascicularis; genus=Macaca (Latin) orMacaque (English); species=fascicularis) and five rhesus (Macacamulatta) monkeys were obtained. 4G7 stability+affinity improved HybridS239D/I332E anti-CD19, anti-CD19 IgG1 (reduced immunogenicity, butwithout affinity/stability optimized variable region), rituximab(anti-CD20), and negative control (enhanced Fc, non-binding variableregion) were directly labeled with FITC. Rituximab was also labeled withAPC to identify the B-cell fraction of cells. Human PBMCs were used aspositive controls throughout. Blood samples and PBMCs were pre-incubatedwith 2 mg/mL of an isotype control antibody with enhanced Fc to blockany potential FcγR binding. In each experiment, rituximab-APC and one ofthe text variants were included in the assay. Detection is made using aFACS Canto II flow cytometer with gate lymphocyte fractions based on theforward and side scattering. Results are shown in FIG. 25.Non-affinity/stability matured anti-CD19 (as well as its parental murineantibody) does not cross-react with cynomolgus or rhesus CD19. Variantsthat increased binding and stability of the anti-CD19 molecule enabledcross-reactivity of 4G7 stability and affinity improved HybridS239D/I332E to both cynomolgus and rhesus CD19.

Example 12. ADCC of an Enhanced Effector Function Anti-CD19 Antibodywith Reduced Fucose Content

Anti-CD19 antibodies with enhanced effector function (4G7 H1L1 HybridS239D/I332E) were evaluated with reduced fucose content. The Lec13 cellline (Ripka et al. Arch. Biochem. Biophys. 49:533-545 (1986)) wasutilized to express anti-CD19 antibodies with reduced fucose content.Lec13 refers to the lectin-resistant Chinese Hamster Ovary (CHO) mutantcell line which displays a defective fucose metabolism and therefore hasa diminished ability to add fucose to complex carbohydrates. That cellline is described in Ripka & Stanley, 1986, Somatic Cell & Molec. Gen.12(1):51-62; and Ripka et al., 1986, Arch. Biochem. Biophys.249(2):533-545. Lec13 cells are believed to lack the transcript forGDP-D-mannose-4,6-dehydratase, a key enzyme for fucose metabolism.Ohyama et al., 1988, J. Biol. Chem. 273(23):14582-14587.GDP-D-mannose-4,6-dehydratase generatesGDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which is thenconverted by the FX protein to GDP-L-fucose. Expression of fucosylatedoligosaccharides is dependent on the GDP-L-fucose donor substrates andfucosyltransferase(s). The Lec13 CHO cell line is deficient in itsability to add fucose, but provides IgG with oligosaccharide which isotherwise similar to that found in normal CHO cell lines and from humanserum (Jefferis, R. et al., 1990, Biochem. J. 268, 529-537; Raju, S. etal., 2000, Glycobiology, 10, 477-486; Routier, F. H., et al., 1997,Glycoconj. J. 14, 201-207). Normal CHO and HEK293 cells add fucose toIgG oligosaccharide to a high degree, typically from 80-98%, and IgGsfrom sera are also highly fucosylated (Jefferis, R. et al., 1990,Biochem. J. 268, 529-537; Raju, S. et al., 2000, Glycobiology 10,477-486; Routier, F. H., et al., 1997, Glycoconj. J. 14, 201-207;Shields et al., 2002, J Biol Chem 277(90):26733-26740). It is wellestablished that antibodies expressed in transfected Lec13 cellsconsistently produce about 10% fucosylated carbohydrate (Shields et al.,2002, J Biol Chem 277(90):26733-26740).

ADCC assays were performed on RS4;11 and MEC-1 cells using anti-CD19antibodies with and without enhanced effector function variants and withand without reduced fucosylation FIG. 26 shows the results of these ADCCassays. Both ADCC potency and efficacy are similar for anti-CD19antibody with amino acid modifications(4G7_H1L1_Hybrid_239D/I332E+fucose) and anti-CD19 IgG1 with reducedfucose content (4G7_H1L1_IgG1_WT−fucose). ADCC potency is furtherincreased by combining amino acid modification with reduced fucosecontent (4G7_H1L1_Hybrid_239D/332E−fucose). (FIG. 26). This experimentthus illustrates that combinations of amino acid modifications andmodified glycoforms may be used to optimize anti-CD19 antibodies foreffector function properties.

The use of the Lec13 cell line is not meant to limit the presentinvention to that particular mode of reducing fucose content. A varietyof other methods are known in the art for controlling the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, including but not limited to expression invarious organisms or cell lines, engineered or otherwise (for exampleLec13 CHO cells or rat hybridoma YB2/0 cells), regulation of enzymesinvolved in the glycosylation pathway (for example FUT8[α],6-fucosyltranserase) and/or β1-4-N-acetylglucosaminyltransferase III[GnTIII]), and modification of modifying carbohydrate(s) after the IgGhas been expressed (Umaña et al., 1999, Nat Biotechnol 17:176-180;Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002,J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem278:3466-3473; Yamane-Ohnuki et al., 2004, Biotechnology andBioengineering 87(5):614-621); (U.S. Pat. No. 6,602,684; U.S. Ser. No.10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO01/29246A1; PCT WO 02/31140A1; PCT WO 02/30954A1).

The use of particular modifications to enhance effector function, forexample the substitutions 239D and 332E and the reduced level of fucose,are not meant to constrain the anti-CD19 antibodies to these particularmodifications. As described above in the section entitled “Modificationsfor optimizing effector function”, a large number of modifications,including amino acid modifications and modified glycoforms, arecontemplated for anti-CD19 antibodies to improve their effector functionproperties.

Example 13. Anti-CD19 Antibodies Inhibit Proliferation of Primary BCells—Applications of Anti-CD19 Antibodies to Treat Autoimmune Diseases

The ability of the anti-CD19 antibodies of this invention to deplete Bcells through ADCC effector function is exemplified by their ability tolyse a variety of cell lines representative of a range of B celllineages, as shown in the preceding examples. This function is mediatedby effector cells such as NK cells and macrophages that express FcγRs,triggering of which induces lysis of the CD19-coated target cells. Anadditional mechanism of action may also be mediated againstantigen-activated B cells. Antigen activation of B cells can be mimickedby the use of antibodies to the B-cell receptor (BCR). This leads totheir proliferation in culture, a generic measure of activation.

Antigen binding can be mimicked in vitro by cross-linking BCR (mu orIgM) with anti-mu (anti-μ, anti-IgM) antibody. In order to demonstratethis activity, Peripheral Blood Mononuclear Cells (PBMCs) were preparedfrom Leukophoresis Pack by Ficoll density gradient, and primary human Bcells were purified from PBMCs using magnetic negative selection kitpurchased from Miltenyi Biotec. The proliferation assay was performed in10% FBS/RPMI1640 medium in total of 100 ul volume in 96 well micro-titerplates in triplicates. B cell activation was induced using F(ab′)2fragment of goat anti-mu antibody (Jackson Immunoresearch, Inc.). In 50ul of medium, serial dilutions of the anti-mu antibody was aliquoted in96 well micro-titer plate, to which 83,000 purified B cells were addedin 50 ul volume. Then the micro-titer plate was incubated at 37° C. for3 days after which, ATP luminescence assay format (Cell TiterGlo Kitfrom Promega) was used to detect the live cells using luminometer. FIG.30a shows that there is a dose-dependence of B cell proliferation onanti-mu antibody concentration.

In order to evaluate the capacity of the WT (4G7_H3_L1 IgG1_WT) andvariant (4G7_H3_L1_Hybrid_239D/332E) anti-CD19 antibodies to modulateB-cell proliferation, an assay was carried out to monitor viability ofprimary human B cells in the presence of anti-CD19 and co-stimulatoranti-mu antibody. As described above, PBMCs were prepared fromLeukophoresis Pack by Ficoll density gradient, and primary human B cellswere purified form PBMCs using magnetic negative selection. Theproliferation assay was performed in 10% FBS/RPMI1640 medium in total of100 ul volume in 96 well micro-titer plates in triplicates. To induceactivation of B cells, the F(ab′)2 fragment of goat anti-mu antibody wasused. In 50 ul of medium, a fixed concentration (2 mg/ml) of anti-muwith five fold serial dilutions of the antibodies were performed in 96well micro-titer plate, to which 100,000 purified B cells were added in50 ul volume. Then the micro-titer plate was incubated at 37° C. for 3days after which, ATP luminescence assay format was used to detect thelive cells using luminometer.

The results, provided in FIG. 30b , show that WT anti-CD19 antibody hasno effect on primary B-cell proliferation, similar to negative controlwith anti-CD30 antibody (CD30 is not expressed on B cells). In contrast,the anti-CD19 antibody comprising Fc modifications has significantinhibitory activity against B-cell viability. Notably, in vitrosignaling as a result of anti-mu antibody cross-linking mimicks antigenengagement of BCR, and is proxy for BCR engagement by autoantigen in aclinical autoimmune setting.

The pathogenesis of most autoimmune diseases is coupled to theproduction of autoantibodies against self antigens, leading to a varietyof associated pathologies. For example, SLE is characterized byproduction of auto- or self-antibodies to double stranded DNA.Accordingly, in the aforedescribed experiment BCR engagement in vitro byanti-mu antibody mimicks stimulation of B cells in lupus patients invivo by anti-double-stranded DNA antibodies. Autoantibodies are producedby terminally differentiated plasma cells that are derived from naïve ormemory B cells. Furthermore, B cells can have other effects onautoimmune pathology, as antigen-presenting cells (APCs) that caninteract with and stimulate helper T cells, further stimulating thecycle of anti-self immune response. Given the expression of CD19 on mostof the B-cell lineage, ranging from pre-B to plasma cells, theantibodies of this invention may have broad utility for the treatment ofautoimmune diseases. Examples of such autoimmune diseases include, butare not limited to, rheumatoid arthritis (RA), systemic lupuserythematosus (SLE or lupus), multiple sclerosis, Sjogren's syndrome,and idiopathic thrombocytopenia purpura (ITP).

The current example demonstrates that anti-CD19 antibodies of theinvention can substantially inhibit B cell proliferation in adose-dependent manner, indicating that they can inhibitantigen-stimulated activation of B cells. B-cell activation by antigencan also initiate the process of class-switching and ultimately terminaldifferentiation into antibody-secreting plasma cells. The antibodies ofthis invention are thus capable of inhibiting these processes via anadditional mechanism of action that does not require effector cells.This inhibition is expected to have beneficial impact on autoimmunedisease by preventing the terminal differentiation of naïve and memory Bcell populations, thus preventing the differentiation ofautoantibody-secreting plasma cells. It is possible that additionalaspects of B-cell biology such as antigen presentation will be affectedby the anti-CD19 antibodies.

> IgG1 G1m(a, z) allotype (SEQ ID NO: 80)     ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > IgG1 G1m(a, x, z) allotype(SEQ ID NO: 81)     ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > IgG1 G1m(f) allotype(SEQ ID NO: 82)     ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > IgG1 G1m(a, f) allotype(SEQ ID NO: 83)     ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > IgG2 G2m(n+) allotype(SEQ ID NO: 84)     ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > IgG2 G2m(n−) allotype (SEQ ID NO: 85)     ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > 4G7 H1 Hybrid S239D/I332E(SEQ ID NO: 86)     EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGSRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > 4G7 H1.52 Hybrid S239/I332E(SEQ ID NO: 87)     EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >4G7 H1.78 Hybrid S239D/I332E (SEQ ID NO: 88)     EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >4G7 H1.191 Hybrid S239D/I332E (SEQ ID NO: 89)     EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >4G7 H1.192 Hybrid S239D/I332E (SEQ ID NO: 90)     EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >4G7 H1.196 Hybrid S239D/I332E (SEQ ID NO: 91)     EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >4G7 H1.201 Hybrid S239D/I332E (SEQ ID NO: 92)     EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNSGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >4G7 H1.202 Hybrid S239D/I332E (SEQ ID NO: 93)     EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNEGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >4G7 H1.203 Hybrid S239D/I332E (SEQ ID NO: 94)     EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNSGTEYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >4G7 H1.204 Hybrid S239D/I332E (SEQ ID NO 95)     EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNEGTEYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > 4G7 L1 (SEQ ID NO: 96)     DIVMTQSPATLSLSPGERATLSCRSSKSLLNSNGNTYLYWFQQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPFTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.26 (SEQ ID NO: 97)     DIVMTQSPATLSLSPGERATLSCRSSKSLLNSNGNTYLYWFQQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPFTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.32(SEQ ID NO: 98)     DIVMTQSPATLSLSPGERATLSCRSSKSLLNSNGNTYLYWFQQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPFTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.64(SEQ ID NO: 99)     DIVMTQSPATLSLSPGERATLSCRSSKSLLNSNGNTYLYWFQQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPFTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.68 (SEQ ID NO: 100)     DIVMTQSPATLSLSPGERATLSCRSSKSLLNSNGNTYLYWFQQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPFTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.96(SEQ ID NO: 101)     DIVMTQSPATLSLSPGERATLSCRSSKSLLNSNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPFTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.145(SEQ ID NO: 102)     DIVMTQSPATLSLSPGERATLSCRSSKSLQNSNGNTYLYWFQQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.148(SEQ ID NO: 103)     DIVMTQSPATLSLSPGERATLSCRSSKSLQNSNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPNTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.49(SEQ ID NO: 104)     DIVMTQSPATLSLSPGERATLSCRSSKSLQNSNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.154(SEQ ID NO: 105)     DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPNTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.155(SEQ ID NO: 106)     DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.160(SEQ ID NO: 107)     DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNANTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.162(SEQ ID NO: 108)     DIVMTQSPATLSLSPGERATLSCRSSKSLQNANANTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.163(SEQ ID NO: 109)     DIVMTQSPATLSLSPGERATLSCRSSKSLQNANSNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 L1.164(SEQ ID NO: 110)     DIVMTQSPATLSLSPGERATLSCRSSKSLQNANSNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > 4G7 VH CDR2 D55A(SEQ ID NO: 111) YINPYNAGTKYNEKFKG > 4G7 VH CDR2 T57P (SEQ ID NO: 112)YINPYNAGTKYNEKFKG > 4G7 VH CDR K58E (SEQ ID NO: 113) YINPYNAGTKYNEKFKG >4G7 VH CDR2 D55S (SEQ ID NO: 114) YINPYNAGTKYNEKFKG > 4G7 VH CDR2 D55E(SEQ ID NO: 115) YINPYNAGTKYNEKFKG > 4G7 VH CDR3 S100T (SEQ ID NO: 116)GTYYYGTRVFDY > 4G7 VH CDR3 R100dS (SEQ ID NO: 117) GTYYYGSSVFDY >4G7 VH CDR3 S100cT/R100dS (SEQ ID NO: 118) GTYYYGTSVFDY >4G7 VL CDR1 L27cQ (SEQ ID NO: 119) RSSKSLQNSNGNTYLY >4G7 VL CDR1 L27cQ/S27eV (SEQ ID NO: 120) RSSKSLQNVNGNTYLY >4G7 VL CDR1 S27eV (SEQ ID NO: 121) RSSKSLQNVNGNTYLY > 4G7 VL CDR1 G29A(SEQ ID NO: 122) RSSKSLLNSNANTYLY > 4G7 VL CDR1 L27eQ/S27eV/G29A(SEQ ID NO: 123) RSSKSLQNVNANTYLY > 4G7 VL CDR1 S27eA (SEQ ID NO: 124)RSSKSLLNANGNTYLY > 4G7 VL CRD1 L27cQ/S27eA/G29A (SEQ ID NO: 125)RSSKSLQNANANTYLY > 4G7 VL CDR1 G29S (SEQ ID NO: 126) RSSKSLLNSNSNTYLY >G7 VL CDR1 L27cQ/S27eA/G29S (SEQ ID NO: 127) RSSKSLQNANSNTYLY >4G7 VL CDR1 L27cQ/S27eA (SEQ ID NO: 128) RSSKSLQNANGNTYLY >4G7 VL CDR2 A55N (SEQ ID NO: 129) RMSNLNS > 4G7 VL CDR3 F96I(SEQ ID NO: 130) MQHLEYPIT > 4G7 VL CDR3 F96N (SEQ ID NO: 131)MQHLEYPNT > 4G7 VH CDR1 (SEQ ID NO: 132): SYVMH >4G7 VH CDR2 (SEQ ID NO: 133): YINPYNDGTKYNEKFKG >4G7 VH CDR3 (SEQ ID NO: 134): GTYYYGSRVFDY >4G7 VL CDR1 (SEQ ID NO: 135): RSSKSLLNSNGNTYLY >4G7 VL CDR2 (SEQ ID NO: 136): RMSNLAS > 4G7 VL CDR3 (SEQ ID NO: 137):MQHLEYPFT > HD37 VH CDR1 (SEQ ID NO: 138): SYWMN >HD37 VH CDR2 (SEQ ID NO: 139): QIWPGDGDTNYNGKFKG >HD37 VH CDR3 (SEQ ID NO: 140): RETTTVGRYYYAMDY >HD37 VL CDR1 (SEQ ID NO: 141): KASQSVDYDGDSYLN >HD37 VL CDR2 (SEQ ID NO: 142): DASNLVS > HD37 VL CDR3 (SEQ ID NO: 143):QQSTEDPWT

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

The invention claimed is:
 1. A method of treating a disease or disorderselected from the group consisting of non-Hodgkin's lymphomas (NHL),chronic lymphocytic leukemia (CLL), B-cell acute lymphoblasticleukemia/lymphoma (B-ALL), and mantle cell lymphoma (MCL), wherein saidmethod comprises administering an antibody that binds CD19, saidantibody comprising a heavy chain and a light chain, said heavy chaincomprising a variable region having a CDR1 comprising SEQ ID NO:132, aCDR2 comprising SEQ ID NO:147, and a CDR3 comprising SEQ ID NO:116; andsaid light chain comprising a variable region having a CDR1 comprisingSEQ ID NO:120, a CDR2 comprising SEQ ID NO:129, and a CDR3 comprisingSEQ ID NO:130, wherein the heavy chain comprises an Fc domain comprisingan amino acid substitution at position S239 and/or I332, wherein the Fcnumbering is according to the EU index as in Kabat.
 2. The method oftreating a disease or disorder according to claim 1, wherein the diseaseor disorder is non-Hodgkin's lymphomas (NHL).
 3. The method of treatinga disease or disorder according to claim 1, wherein the disease ordisorder is chronic lymphocytic leukemia (CLL).
 4. The method oftreating a disease or disorder according to claim 1, wherein the diseaseor disorder is B-cell acute lymphoblastic leukemia/lymphoma (B-ALL). 5.The method of treating a disease or disorder according to claim 1,wherein the disease or disorder is mantle cell lymphoma (MCL).
 6. Themethod of treating a disease or disorder according to claim 1, whereinsaid heavy chain variable region comprises SEQ ID NO:40.
 7. The methodof treating a disease or disorder according to claim 1, wherein saidlight chain variable region comprises SEQ ID NO:58.
 8. The method oftreating a disease or disorder according to claim 1, wherein said heavychain comprises SEQ ID NO:40 and said light chain sequence comprises SEQID NO:58.
 9. The method of treating a disease or disorder according toclaim 1, wherein the amino acid substitution is at position S239. 10.The method of treating a disease or disorder according to claim 6,wherein the amino acid substitution is at position S239.
 11. The methodof treating a disease or disorder according to claim 7, wherein theamino acid substitution is at position S239.
 12. The method of treatinga disease or disorder according to claim 8, wherein the amino acidsubstitution is at position S239.
 13. The method of treating a diseaseor disorder according to claim 1, wherein the amino acid substitution isat position I332.
 14. The method of treating a disease or disorderaccording to claim 6, wherein the acid substitution is at position I332.15. The method of treating a disease or disorder according to claim 7,wherein the amino acid substitution is amino acid substitution atposition I332.
 16. The method of treating a disease or disorderaccording to claim 8, wherein the amino acid substitution is at positionI332.
 17. The method of treating a disease or disorder according toclaim 1, wherein the amino acid substitutions are S239D and I332E. 18.The method of treating a disease or disorder according to claim 6,wherein the amino acid substitutions are S239D and I332E.
 19. The methodof treating a disease or disorder according to claim 7, wherein theamino acid substitutions are S239D and I332E.
 20. The method of treatinga disease or disorder according to claim 8, wherein the amino acidsubstitutions are S239D and I332E.
 21. The method of treating a diseaseor disorder according to claim 1, wherein said method comprisesadministering a composition comprising a plurality of glycosylatedantibodies that bind CD19, said antibody comprising a heavy chain and alight chain, said heavy chain comprising a variable region having a CDR1comprising SEQ ID NO:132, a CDR2 comprising SEQ ID NO:147, and a CDR3comprising SEQ ID NO:116; and said light chain comprising a variableregion having a CDR1 comprising SEQ ID NO:120, a CDR2 comprising SEQ IDNO:129, and a CDR3 comprising SEQ ID NO:130, wherein about 80-100% ofthe glycosylated antibodies in the composition comprise a mature corecarbohydrate structure which lacks fucose, and wherein the heavy chaincomprises an Fc domain comprising an amino acid substitution at positionS239 and/or I332, the Fc numbering is according to the EU index as inKabat.
 22. The method of treating a disease or disorder according toclaim 1, wherein said heavy chain variable region comprises SEQ ID NO:87.
 23. The method of treating a disease or disorder according to claim1, wherein said light chain variable region comprises SEQ ID NO: 106.24. The method of treating a disease or disorder according to claim 1,wherein said heavy chain variable region comprises SEQ ID NO: 87 andsaid light chain variable region comprises SEQ ID NO: 106.